Variants of Adrenomedullin and Calcitonin Gene-Related Peptide and Methods of Use

ABSTRACT

Variant peptides of calcitonin gene-related peptide alpha (αCGRP), calcitonin gene-related peptide beta (βCGRP), and adrenomedullin (AM) are disclosed, wherein the variant peptides have high binding affinity and agonistic or antagonistic activity for at least one receptor complex of CLR:RAMP1, CLR:RAMP2, and CLR:RAMP3. Also disclosed are methods of use of the variant peptides in therapeutic treatments.

CROSS REFERENCE TO RELATED APPLICATIONS/ INCORPORATION BY REFERENCESTATEMENT

This application claims benefit under 35 USC § 119(e) of ProvisionalApplication U.S. Ser. No. 62/160,899, filed May 13, 2015, the entiretyof which is hereby expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support from National Institutesof Health (NIH) grant R01GM104251. The government has certain rights inthe invention.

BACKGROUND

G protein-coupled receptors (GPCRs) are a large family of cell surfacereceptors that regulate a multitude of biological processes in responseto a diverse array of stimuli, and they are important drug targets. Theclass B/Secretin family of GPCRs in humans includes fifteen receptorsthat are activated by diverse neuropeptides, peptide paracrine factorsand peptide endocrine hormones. Class B GPCRs comprise an extracellulardomain (ECD) of about 120 amino acids in addition to a 7-transmembrane(7TM) domain in the membrane. The ECD has an N-terminal α-helix and aset of β-sheets held together by three disulfide bonds. Activatingpeptides bind class B GPCRs via a “two-domain” model, whereby theC-terminal region of the peptide binds the ECD, and the N-terminalregion of the peptide binds and activates the 7TM domain. Certain of theactivating peptides bind as extended α-helices to the same region of thereceptor, in a groove between the N- and C-termini of the isolated ECDs.However, the mechanism of binding of the calcitonin (CT) family of thisgroup of Class B GPCR-activating peptides has been unknown.

Members of the CT family of activating peptides include, for example,calcitonin gene-related peptide alpha (αCGRP), calcitonin gene-relatedpeptide beta (βCGRP), adrenomedullin (AM), adrenomedullin 2/intermedin(AM2), amylin (Amy), and CT. These C-terminally amidated peptides have arange of actions including neurogenic inflammation, e.g., as a factor inmigraine headache pathogenesis (CGRP), vasodilation/cardioprotection(CGRP, AM and AM2), and regulation of blood and lymphatic vasculardevelopment (AM), nutrient intake and blood glucose (Amy), and boneturnover (CT). CGRP in the trigeminovascular system acts as a painsignaling neurotransmitter. Elevated CGRP levels are associated withmigraines, and exogenous administration of CGRP to migraineurs triggersmigraines. Small molecule antagonists of the CGRP receptor that bind theECD complex showed efficacy in clinical trials, but were abandonedbecause of toxicity unrelated to CGRP signaling.

Binding of CGRP, AM, and AM2 to their cognate class B receptor, thecalcitonin receptor-like receptor (CLR), is dependent on association ofCLR with one of three accessory membrane proteins that determine ligandselectivity: receptor activity-modifying proteins (RAMPs) 1, 2, or 3.RAMPs have an ECD of about 100 amino acids and a single TM segment.CLR:RAMP1 is a CGRP receptor, CLR:RAMP2 preferentially recognizes AM andis called the AM₁ receptor, and CLR:RAMP3 binds both AM and AM2 withhigh affinities and is called the AM₂ receptor. Amy has a low affinityfor the class B CT receptor (CTR); however, when CTR associates with anyof the RAMPs, its affinity for Amy is markedly increased. CTR alone isthe receptor for CT. Thus the RAMPs profoundly alter the behavior of CLRand CTR.

For peptide-based therapeutics targeting the CGRP or AM receptors,selectivity against the CTR and AMY receptors is desired because oftheir different functions. CTR is involved in calcium homeostasis andbone remodeling. Salmon CT, which has higher affinity for human CTR thanhuman CT, has long been used as a therapeutic for Paget's disease andosteoporosis. Amy signaling controls blood glucose levels and regulatesfood intake. The Amy analog Pramlintide is used as an insulin adjuncttherapy for types I and II diabetes, and AMY activation is explored asan obesity treatment.

Crystal structures have been available for ligand-free and smallmolecule antagonist-bound CLR:RAMP1 and ligand-free CLR:RAMP2 ECDcomplexes, but these have provided little insight into how the peptidesbind or how RAMPs determine selectivity. In a further complication, ithas appeared unlikely that CGRP and AM bind as extended helices as seenwith other class B peptide ligands; there is evidence that only a smallportion of these peptides form α-helices and that at their C-termini,there are one or more turn structures. A better understanding of themechanism of binding of CGRP and AM to their respective CLR:RAMPreceptor complexes has been desired.

BRIEF DESCRIPTION OF THE DRAWINGS

Several embodiments of the inventive concepts of the present disclosureare illustrated in the appended drawings. It is to be noted, however,that the appended drawings only illustrate several embodiments and aretherefore not intended to be considered limiting of the scope of thepresent disclosure.

FIG. 1 depicts in (A) binding of a CGRP C-terminal peptide,CGRP(8-37)NH₂, and two variant AM C-terminal peptides(AM(37-52)NH₂[Q50W] and AM(37-52)NH₂[S45W, Q50W]) to purified CGRPreceptor extracellular domain complex (CLR:RAMP1). (B) depicts bindingof an AM C-terminal peptide, AM(37-52)NH₂, and the two variant AMC-terminal peptides to purified AM₁ receptor extracellular domaincomplex (CLR:RAMP2). Single representative experiments conducted withduplicate samples are shown. Error bars are S.E.M.

FIG. 2(A-C) depicts binding of (A) AM-based peptide AM(37-52)NH₂, (B)variant peptides AM(37-52)NH₂[S45W, Q50W], AM(37-52)NH₂[Q50F], andAM(37-52)NH₂[Q50W], and (C) variant peptides AM(37-52)NH₂[D39K],AM(37-52)NH₂[K46L, Q50W], and AM(37-52)NH₂[K46L, Q50W, Y52F], topurified CGRP receptor extracellular domain complex. Singlerepresentative experiments conducted with duplicate samples are shown.Error bars are S.E.M.

FIG. 2(D-E) depicts binding of (D) variant peptides AM(37-52)NH₂[D39F,K46L, Q50W], AM(37-52)NH₂[D39F, K46L, Q50W, Y52F], andAM(37-52)NH₂[S45W, K46L, Q50W, Y52F], and (E) variant peptidesAM(37-52)NH₂[S45W, K46M, Q50W, Y52F], and AM(37-52)NH₂[S45W, K46W, Q50W,Y52F], to purified CGRP receptor extracellular domain complex. Singlerepresentative experiments conducted with duplicate samples are shown.Error bars are S.E.M.

FIG. 2(F-H) depicts binding of (F) variant peptides AM(37-52)NH₂[S45T,K46W, Q50W], AM(37-52)NH₂[S45T, K46L, Q50W], and AM(37-52)NH₂[S45W,K46L, S48G, Q50W, Y52F], (G) variant peptide AM(37-52)NH₂[S45W, K46L,I47M, Q50W, Y52F], and (H) variant peptides AM(37-52)NH₂[S45R, K46W,Q50W], and AM(37-52)NH₂[S45R ,K46L, Q50W], to purified CGRP receptorextracellular domain complex. Single representative experimentsconducted with duplicate samples are shown. Error bars are S.E.M.

FIG. 2(I-J) depicts binding of (I) variant peptides AM(37-52)NH₂[S45R,K46W, S48G, Q50W], AM(37-52)NH₂[S45R, K46L, S48G, Q50W], andAM(37-52)NH₂[S45R, K46W, I47L, S48G, Q50W], and (J) variant peptidesAM(37-52)NH₂[D39W, Q50W], and AM(37-52)NH₂[D37V, S45W, K46L, Q50W,Y52F], to purified CGRP receptor extracellular domain complex. Singlerepresentative experiments conducted with duplicate samples are shown.Error bars are S.E.M.

FIG. 3(A-C) depicts binding of (A) AM-based peptide AM(37-52)NH₂, andvariant peptide AM(37-52)NH₂[S45W, Q50W], (B) AM-based peptideAM(37-52)NH₂, variant peptide AM(37-52)NH₂[Q50F], and variant peptideAM(37-52)NH₂[Q50W], and (C) variant peptides AM(37-52)NH₂[K46L],AM(37-52)NH₂[K46M], and AM(37-52)NH₂[D39F], to purified AM₁ receptorextracellular domain complex. Single representative experimentsconducted with duplicate samples are shown. Error bars are S.E.M.

FIG. 3(D-F) depicts binding of (D) variant peptides AM(37-52)NH₂[D39F,K46L, Q50W], AM(37-52)NH₂[D39F, K46L, Q50W, Y52F], andAM(37-52)NH₂[S45W, K46L, Q50W, Y52F], (E) variant peptidesAM(37-52)NH₂[[D39K], AM(37-52)NH₂[K46L, Q50W], and AM(37-52)NH₂[K46L,Q50W, Y52F], and (F) variant peptides AM(37-52)NH₂[S45W, K46M, Q50W,Y52F], and AM(37-52)NH₂[S45W, K46W, Q50W, Y52F], to purified AM₁receptor extracellular domain complex. Single representative experimentsconducted with duplicate samples are shown. Error bars are S.E.M.

FIG. 3(G-H) depicts binding of (G) variant peptides AM(37-52)NH₂[S45T,K46W, Q50W], AM(37-52)NH₂[S45T, K46L, Q50W], and AM(37-52)NH₂[S45W,K46L, S48G, Q50W, Y52F], and (H) variant peptide AM(37-52)NH₂[S45W,K46L, I47M, Q50W, Y52F], to purified AM₁ receptor extracellular domaincomplex. Single representative experiments conducted with duplicatesamples are shown. Error bars are S.E.M.

FIG. 3(I-K) depicts binding of (I) variant peptides AM(37-52)NH₂[S45R,K46W, Q50W], and AM(37-52)NH₂[S45R, K46L, Q50W], (J) variant peptidesAM(37-52)NH₂[S45R, K46W, S48G, Q50W], AM(37-52)NH₂[S45R, K46L, S48G,Q50W], and AM(37-52)NH₂[S45R, K46W, I47L, S48G, Q50W], and (K) variantpeptides AM(37-52)NH₂[D39W, Q50W], and AM(37-52)NH₂[D37V, S45W, K46L,Q50W, Y52F], to the purified AM₁ receptor extracellular domain complex.Single representative experiments conducted with duplicate samples areshown. Error bars are S.E.M.

FIG. 4(A-C) depicts binding of (A) CGRP-based peptide CGRP[8-37]NH₂, (B)variant peptides CGRP(27-37)NH₂[N31D, S34P, K35F], CGRP(27-37)NH₂[N31D,S34P, K35W], and CGRP(27-37)NH₂[N31D, S34P, K35F, A36S], and (C) variantpeptides CGRP(27-37)NH₂[N31D, S34P, K35W, A36S], andCGRP(27-37)NH₂[N31D, S34P, K35W, A36S, F37Y], to purified CGRP receptorextracellular domain complex. Single representative experimentsconducted with duplicate samples are shown. Error bars are S.E.M.

FIG. 4(D-E) depicts binding of (D) variant peptides CGRP(27-37)NH₂[N31D,V32T, S34P, K35W, A36G], and CGRP(27-37)NH₂[N31D, V32T, S34P, K35W,A36G, F37Y], and (E) variant peptide CGRP(27-37)NH₂[N31D, V32T, S34P,K35W, A36S], to purified CGRP receptor extracellular domain complex.Single representative experiments conducted with duplicate samples areshown. Error bars are S.E.M.

FIG. 5(A-C) depicts binding of (A) CGRP-based peptide CGRP(11-37)NH₂,(B) variant peptides CGRP(27-37)NH₂[N31D, S34P, K35F],CGRP(27-37)NH₂[N31D, S34P, K35W], and CGRP(27-37)NH₂[N31D, S34P, K35F,A36S], and (C) variant peptides CGRP(27-37)NH₂[N31D, S34P, K35W, A36S],and CGRP(27-37)NH₂[N31D, S34P, K35W, A36S, F37Y], to the purified AM₁receptor extracellular domain complex. Single representative experimentsconducted with duplicate samples are shown. Error bars are S.E.M.

FIG. 5(D-E) depicts binding of (D) variant peptides CGRP(27-37)NH₂[N31D,V32T, S34P, K35W, A36G], and CGRP(27-37)NH₂[N31D, V32T, S34P, K35W,A36G, F37Y], and (E) variant peptide CGRP(27-37)NH₂[N31D, V32T, S34P,K35W, A36S], to purified AM₁ receptor extracellular domain complex.Single representative experiments conducted with duplicate samples areshown. Error bars are S.E.M.

FIG. 6 depicts binding of CGRP variant peptide CGRP(27-37)NH₂ [N31D,S34P, K35W, A36S] and AM variant peptide AM(37-52)NH₂ [S45W, K46L, Q50W,Y52F], to purified AMY₁ receptor extracellular domain complex. A singlerepresentative experiment conducted with duplicate samples is shown.Error bars are S.E.M.

FIG. 7(A-B) depicts antagonism of cAMP signaling at full-length (A) CGRP(RAMP1:CLR) and (B) AM₁ (RAMP2:CLR) receptor complexes transientlyexpressed in COS-7 cells by the variant peptideAM(37-52)NH₂[S45W/K46L/Q50W/Y52F]. A single representative experimentconducted with duplicate samples is shown. Error bars are S.E.M.

FIG. 8(A-F) depicts binding of AM-based PS-SPCL library mixtures topurified CGRP (A, C, E) and AM₁ (B, D, F) receptor extracellular domaincomplexes. One of 5 positions of the 16 positions in variantAM(37-52)NH₂[Q50W] was optimized for each mixture: (A, B) position 45optimized; (C, D) position 46 optimized; and (E, F) position 47optimized. Single experiments conducted with duplicate samples areshown. Error bars are S.E.M. (−) indicates no competitor control.

FIG. 8(G-J) depicts binding of AM-based PS-SPCL library mixtures topurified CGRP (G, I) and AM₁ (H, J) receptor extracellular domaincomplexes. One of 5 positions of the 16 positions in variantAM(37-52)NH₂[Q50W] was optimized for each mixture: (G, H) position 48optimized; and (I, J) position 52 optimized. Single experimentsconducted with duplicate samples are shown. Error bars are S.E.M. (−)indicates no competitor control.

FIG. 9(A-C) depicts binding of CGRP-based PS-SPCL library mixtures topurified CGRP receptor extracellular domain complexes. One of 5positions of the 11 positions in variant CGRP(27-37)NH₂[S34P, K35W] wasoptimized for each mixture: (A) position 31 optimized; (B) position 32optimized; and (C) position 33 optimized. Single experiments conductedwith duplicate samples are shown. Error bars are S.E.M. (−) indicates nocompetitor control.

FIG. 9(D-E) depicts binding of CGRP-based PS-SPCL library mixtures topurified CGRP receptor extracellular domain complexes. One of 5positions of the 11 positions in variant CGRP(27-37)NH₂[S34P, K35W] wasoptimized for each mixture: (D) position 36 optimized; and (E) position37 optimized. Single experiments conducted with duplicate samples areshown. Error bars are S.E.M. (−) indicates no competitor control.

DETAILED DESCRIPTION

High-resolution crystal structures of CGRP analog-bound CLR:RAMP1 andAM-bound CLR:RAMP2 ECD heterodimers obtained during the present workrevealed bound peptide conformations starkly different from other classB GPCR peptide ligands and helped explain how RAMPs determine peptideselectivity. This work has enabled a better understanding of themechanism of binding of AM and CGRP to their cognate proteins, enablingthe formulation herein of variants of AM, AM2, and CGRP peptides,including (but not limited to) variant AM, AM2, and CGRP peptidefragments that are able to induce antagonistic or enhanced agonisticactivity in one or more of the CLR:RAMP receptor complexes for use intreatments of various diseases and conditions involving AM and CGRPreceptor proteins. The present disclosure describes such variants andtherapeutic methods of their use.

Before further describing various embodiments of the compounds,compositions, and methods of the present disclosure in more detail byway of exemplary description, examples, and results, it is to beunderstood that the compounds, compositions, and methods of the presentdisclosure are not limited in application to the details of specificembodiments and examples as set forth in the following description. Thedescription provided herein is intended for purposes of illustrationonly and is not intended to be construed in a limiting sense. As such,the language used herein is intended to be given the broadest possiblescope and meaning, and the embodiments and examples are meant to beexemplary, not exhaustive. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting unless otherwiseindicated as so. Moreover, in the following detailed description,numerous specific details are set forth in order to provide a morethorough understanding of the present disclosure. However, it will beapparent to a person having ordinary skill in the art that the presentdisclosure may be practiced without these specific details. In otherinstances, features which are well known to persons of ordinary skill inthe art have not been described in detail to avoid unnecessarycomplication of the description. It is intended that all alternatives,substitutions, modifications, and equivalents apparent to those havingordinary skill in the art are included within the scope of the presentdisclosure. All of the compounds, compositions, and methods ofproduction and application and use thereof disclosed herein can be madeand executed without undue experimentation in light of the presentdisclosure. Thus, while the compounds, compositions, and methods of thepresent disclosure have been described in terms of particularembodiments, it will be apparent to those of skill in the art thatvariations may be applied to the compounds, compositions, and/or methodsand in the steps or in the sequence of steps of the methods describedherein without departing from the concept, spirit, and scope of theinventive concepts.

All patents, published patent applications, and non-patent publicationsmentioned in the specification or referenced in any portion of thisapplication are herein expressly incorporated by reference in theirentirety to the same extent as if each individual patent or publicationwas specifically and individually indicated to be incorporated byreference.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those having ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

As utilized in accordance with the methods and compositions of thepresent disclosure, the following terms, unless otherwise indicated,shall be understood to have the following meanings:

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or when the alternatives are mutually exclusive,although the disclosure supports a definition that refers to onlyalternatives and “and/or.” The use of the term “at least one” will beunderstood to include one as well as any quantity more than one,including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 100, or any integer inclusive therein. The term “at least one”may extend up to 100 or 1000 or more, depending on the term to which itis attached; in addition, the quantities of 100/1000 are not to beconsidered limiting, as higher limits may also produce satisfactoryresults. In addition, the use of the term “at least one of X, Y, and Z”will be understood to include X alone, Y alone, and Z alone, as well asany combination of X, Y, and Z.

As used herein, all numerical values or ranges include fractions of thevalues and integers within such ranges and fractions of the integerswithin such ranges unless the context clearly indicates otherwise. Thus,to illustrate, reference to a numerical range, such as 1-10 includes 1,2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc.,and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., upto and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2,2.3, 2.4, 2.5, etc., and so forth. Reference to a series of rangesincludes ranges which combine the values of the boundaries of differentranges within the series. Thus, to illustrate reference to a series ofranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75,75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750,750-1,000, includes ranges of 1-20, 10-50, 50-100, 100-500, and500-1,000, for example.

As used in this specification and claims, the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the composition, themethod used to administer the composition, or the variation that existsamong the study subjects. As used herein the qualifiers “about” or“approximately” are intended to include not only the exact value,amount, degree, orientation, or other qualified characteristic or value,but are intended to include some slight variations due to measuringerror, manufacturing tolerances, stress exerted on various parts orcomponents, observer error, wear and tear, and combinations thereof, forexample. The term “about” or “approximately,” where used herein whenreferring to a measurable value such as an amount, a temporal duration,and the like, is meant to encompass, for example, variations of ±10%, or±5%, or ±1%, or ±0.1% from the specified value, as such variations areappropriate to perform the disclosed methods and as understood bypersons having ordinary skill in the art. As used herein, the term“substantially” means that the subsequently described event orcircumstance completely occurs or that the subsequently described eventor circumstance occurs to a great extent or degree. For example, theterm “substantially” means that the subsequently described event orcircumstance occurs at least 90% of the time, or at least 95% of thetime, or at least 98% of the time.

As used herein any reference to “one embodiment” or “an embodiment”means that a particular element, feature, structure, or characteristicdescribed in connection with the embodiment is included in at least oneembodiment and may be included in other embodiments. The appearances ofthe phrase “in one embodiment” in various places in the specificationare not necessarily all referring to the same embodiment and are notnecessarily limited to a single or particular embodiment.

The term “pharmaceutically acceptable” refers to compounds andcompositions which are suitable for administration to humans and/oranimals without undue adverse side effects such as toxicity, irritationand/or allergic response commensurate with a reasonable benefit/riskratio. The compounds of the present disclosure may be combined with oneor more pharmaceutically-acceptable excipients, including carriers,vehicles, and diluents which may improve solubility, deliverability,dispersion, stability, and/or conformational integrity of the compoundsor conjugates thereof.

As used herein, “pure,” or “substantially pure” means an object speciesis the predominant species present (i.e., on a molar basis it is moreabundant than any other object species in the composition thereof), andparticularly a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80% of all macromolecularspecies present in the composition, more particularly more than about85%, more than about 90%, more than about 95%, or more than about 99%.The term “pure” or “substantially pure” also refers to preparationswhere the object species is at least 60% (w/w) pure, or at least 70%(w/w) pure, or at least 75% (w/w) pure, or at least 80% (w/w) pure, orat least 85% (w/w) pure, or at least 90% (w/w) pure, or at least 92%(w/w) pure, or at least 95% (w/w) pure, or at least 96% (w/w) pure, orat least 97% (w/w) pure, or at least 98% (w/w) pure, or at least 99%(w/w) pure, or 100% (w/w) pure.

Non-limiting examples of animals within the scope and meaning of thisterm include dogs, cats, rats, mice, guinea pigs, chinchillas, horses,goats, cattle, sheep, zoo animals, Old and New World monkeys, non-humanprimates, and humans.

“Treatment” refers to therapeutic treatments. “Prevention” refers toprophylactic or preventative treatment measures or reducing the onset ofa condition or disease. The term “treating” refers to administering thecomposition to a subject for therapeutic purposes and/or for prevention.Non-limiting examples of modes of administration include oral, topical,retrobulbar, subconjunctival, transdermal, parenteral, subcutaneous,intranasal, intramuscular, intraperitoneal, intravitreal, andintravenous routes, including both local and systemic applications. Theterm “topical” is used herein to define a mode of administration throughan epithelial surface, such as but not limited to, the skin, eye, orinternal epithelial surfaces. In addition, the compositions of thepresent disclosure may be designed to provide delayed, controlled,extended, and/or sustained release using formulation techniques whichare well known in the art.

The terms “therapeutic composition” and “pharmaceutical composition”refer to a composition containing a peptide as described herein that maybe administered to a subject by any method known in the art or otherwisecontemplated herein, wherein administration of the composition bringsabout a therapeutic effect as described elsewhere herein.

The term “effective amount” refers to an amount of a peptide or peptidecompound which is sufficient to exhibit a detectable therapeutic,amelioration, or treatment effect in a subject without excessive adverseside effects (such as substantial toxicity, irritation and allergicresponse) commensurate with a reasonable benefit/risk ratio when used inthe manner of the present disclosure. The effective amount for a subjectwill depend upon the subject's type, size and health, the nature andseverity of the condition to be treated, the method of administration,the duration of treatment, the nature of concurrent therapy (if any),the specific formulations employed, and the like. Thus, it is notpossible to specify an exact effective amount in advance. However, theeffective amount for a given situation can be determined by one ofordinary skill in the art using routine experimentation based on theinformation provided herein.

The term “ameliorate” means a detectable or measurable improvement in asubject's condition or a symptom thereof. A detectable or measurableimprovement includes a subjective or objective decrease, reduction,inhibition, suppression, limit or control in the occurrence, frequency,severity, progression, or duration of the condition, or an improvementin a symptom or an underlying cause or a consequence of the condition,or a reversal of the condition. A successful treatment outcome can leadto a “therapeutic effect,” or “benefit” of ameliorating, decreasing,reducing, inhibiting, suppressing, limiting, controlling or preventingthe occurrence, frequency, severity, progression, or duration of acondition, or consequences of the condition in a subject.

A decrease or reduction in worsening, such as stabilizing the condition,is also a successful treatment outcome. A therapeutic benefit thereforeneed not be complete ablation or reversal of the condition, or any one,most or all adverse symptoms, complications, consequences or underlyingcauses associated with the condition. Thus, a satisfactory endpoint maybe achieved when there is an incremental improvement such as a partialdecrease, reduction, inhibition, suppression, limit, control orprevention in the occurrence, frequency, severity, progression, orduration, or inhibition or reversal of the condition (e.g.,stabilizing), over a short or long duration of time (e.g., seconds,minutes, hours).

The term “homologous” or “% identity” as used herein means a nucleicacid (or fragment thereof) or a protein (or a fragment thereof) having adegree of homology to the corresponding natural reference nucleic acidor protein that may be in excess of 70%, or in excess of 80%, or inexcess of 85%, or in excess of 90%, or in excess of 91%, or in excess of92%, or in excess of 93%, or in excess of 94%, or in excess of 95%, orin excess of 96%, or in excess of 97%, or in excess of 98%, or in excessof 99%. For example, in regard to peptides or polypeptides, thepercentage of homology or identity as described herein is typicallycalculated as the percentage of amino acid residues found in the smallerof the two sequences which align with identical amino acid residues inthe sequence being compared, when four gaps in a length of 100 aminoacids may be introduced to assist in that alignment (as set forth byDayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p. 124,National Biochemical Research Foundation, Washington, D.C. (1972)). Inone embodiment, the percentage homology as described above is calculatedas the percentage of the components found in the smaller of the twosequences that may also be found in the larger of the two sequences(with the introduction of gaps), with a component being defined as asequence of four, contiguous amino acids. Also included as substantiallyhomologous is any protein product which may be isolated by virtue ofcross-reactivity with antibodies to the native protein product. Sequenceidentity or homology can be determined by comparing the sequences whenaligned so as to maximize overlap and identity while minimizing sequencegaps. In particular, sequence identity may be determined using any of anumber of mathematical algorithms. A non-limiting example of amathematical algorithm used for comparison of two sequences is thealgorithm of Karlin & Altschul (Proc. Natl. Acad. Sci. USA (1990)87:2264-2268), modified as in Karlin & Altschul (Proc. Natl. Acad. Sci.USA (1993) 90:5873-5877).

In one embodiment “% identity” represents the number of amino acids ornucleotides which are identical at corresponding positions in twosequences of a protein having the same activity or encoding similarproteins. For example, two amino acid sequences each having 100 residueswill have 95% identity when 95 of the amino acids at correspondingpositions are the same.

Another example of a mathematical algorithm used for comparison ofsequences is the algorithm of Myers & Miller (CABIOS (1988) 4:11-17).Such an algorithm is incorporated into the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used. Yet another useful algorithm for identifying regions oflocal sequence similarity and alignment is the FASTA algorithm asdescribed in Pearson & Lipman (Proc. Natl. Acad. Sci. USA (1988)85:2444-2448).

Another algorithm is the WU-BLAST (Washington University BLAST) version2.0 software (WU-BLAST version 2.0 executable programs for several UNIXplatforms). This program is based on WU-BLAST version 1.4, which in turnis based on the public domain NCBI-BLAST version 1.4 (Altschul & Gish,Methods in Enzymology (1996) 266:460-480; Altschul et al., J Molec Biol.(1990) 215:403-410; Gish & States, Nature Genetics (1993) 3:266-272;Karlin & Altschul, Proc. Natl. Acad. Sci. USA (1993) 90:5873-5877; allof which are incorporated by reference herein).

In addition to those otherwise mentioned herein, mention is made also ofthe programs BLAST, gapped BLAST, BLASTN, BLASTP, and PSI-BLAST,provided by the National Center for Biotechnology Information. Theseprograms are widely used in the art for this purpose and can alignhomologous regions of two amino acid sequences. In all search programsin the suite, the gapped alignment routines are integral to the databasesearch itself. Gapping can be turned off if desired. The default penalty(Q) for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 forBLASTN, but may be changed to any integer. The default per-residuepenalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10for BLASTN, but may be changed to any integer. Any combination of valuesfor Q and R can be used in order to align sequences so as to maximizeoverlap and identity while minimizing sequence gaps. The default aminoacid comparison matrix is BLOSUM62, but other amino acid comparisonmatrices such as PAM can be utilized.

Specific amino acids may be referred to herein by the followingdesignations: alanine: ala or A; arginine: arg or R; asparagine: asn orN; aspartic acid: asp or D; cysteine: cys or C; glutamic acid: glu or E;glutamine: gln or Q; glycine: gly or G; histidine: his or H; isoleucine:ile or I; leucine: leu or L; lysine: lys or K; methionine: met or M;phenylalanine: phe or F; proline: pro or P; serine: ser or S; threonine:thr or T; tryptophan: trp or W; tyrosine: tyr or Y; and valine: val orV.

The terms “polynucleotide sequence” or “nucleic acid,” as used herein,include any polynucleotide sequence which encodes a mutant peptideincluding polynucleotides in the form of RNA, such as mRNA, or in theform of DNA, including, for instance, cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The DNA may be double-stranded or single-stranded.Single-stranded DNA may be the coding strand, also known as the sensestrand, or it may be the non-coding strand, also referred to as theanti-sense strand. The polynucleotide sequence encoding a mutantpeptide, or encoding a therapeutically-effective fragment of a mutantpeptide can be substantially the same as the coding sequence of theendogenous coding sequence as long as it encodes a biologically activemutant peptide. Further, the mutant peptide, ortherapeutically-effective fragment of a mutant peptide may be expressedusing polynucleotide sequence(s) which differ in codon usage due to thedegeneracies of the genetic code or allelic variations. Moreover, themutant peptides of the presently disclosed inventive concepts and thenucleic acids which encode them include peptide and nucleic acidvariants which comprise additional conservative substitutions. Forexample, the variant peptides include, but are not limited to, variantsthat are not exactly the same as the sequences disclosed herein, butwhich have, in addition to the substitutions explicitly described forvarious sequences listed herein, conservative substitutions of aminoacid residues which do substantially not impair the agonistic orantagonistic activity or properties of the variants described herein.Examples of such conservative amino acid substitutions include, but arenot limited to, ala to gly, ser, or thr; arg to gln, his, or lys; asn toasp, gln, his, lys, ser, or thr; asp to asn or glu; cys to ser; gln toarg, asn, glu, his, lys, or met; glu to asp, gln, or lys; gly to pro orala; his to arg, asn, gln, or tyr; ile to leu, met, or val; leu to ile,met, phe, or val; lys to arg, asn, gln, or glu; met to gln, ile, leu, orval; phe to leu, met, trp, or tyr; ser to ala, asn, met, or thr; thr toala, asn, ser, or met; trp to phe or tyr; tyr to his, phe or trp; andval to ile, leu, or met.

Where referred to herein, the terms “variant” and “mutant” are usedinterchangeably. A variant peptide, peptide variant, or mutant refers toany peptide described herein, including fragments thereof, comprising atleast one amino acid substitution in the wild type amino acid sequenceor in a fragment thereof. Non-limiting examples of such variant peptidesare described in further detail below.

Where used herein, the term CLR:RAMP1 refers to the CGRP receptor,CLR:RAMP2 refers to the AM₁ receptor, and CLR:RAMP3 refers to the AM₂receptor.

Where used herein the term “fragment” refers to a portion of a completeamino acid sequence or nucleic acid sequence. For example, a fragment ofwild type AM (SEQ ID NO:1) refers, in at least certain embodiments, to apeptide having 5 or more contiguous amino acids of wild type AM, forexample, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, or 44 amino acids. A fragment of wild type CGRP (SEQ IDNOs: 31 and 32) refers, in at least certain embodiments, to a peptidehaving 4 or more contiguous amino acids of wild type α-CGRP or wild typeβ-CGRP, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 aminoacids. A fragment of wild type AM2/intermedin (SEQ ID NO:47) refers, inat least certain embodiments, to a peptide having 5 or more contiguousamino acids of wild type AM2/intermedin, for example, 5, 6, 7, 8, 9, 10,11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, or 32 amino acids.

Non-limiting embodiments of variant peptides of the present disclosureare included in specific examples shown below, which comprise just asmall subset of the variant peptides supported and enabled herein. Inall specific sequences below except SEQ ID NO:48, the C-terminal residueis amidated (i.e., the C-terminal OH group is replaced by NH₂), e.g., inSEQ ID NOs:1-47 and 49-52 the Y or F is amidated. In SEQ ID NOs: 53 and54, the C-terminal residue is also amidated.

Wild-type human adrenomedullin ((AM(1-52)-NH₂)) is a 52 amino acid,C-terminally-amidated peptide having the sequence:

YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY (SEQ ID NO:1), witha disulfide bond between C16 and C21.

AM (13-52)-NH₂ is a fragment of SEQ ID NO:1 having the sequence:

SFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY (SEQ ID NO:2), with a disulfidebond between the two cysteines.

AM (22-52)-NH₂ is a C-terminal fragment of SEQ ID NO:1 having thesequence:

(SEQ ID NO: 3) TVQKLAHQIYQFTDKDKDNVAPRSKISPQGY.

AM (37-52)-NH₂ is a C-terminal fragment of SEQ ID NO:1 having thesequence:

(SEQ ID NO: 4) DKDNVAPRSKISPQGY.

SEQ ID NOS: 5-30 below are mutants of SEQ ID NO:4 (C-terminal aminoacids 37-52 of SEQ ID NO:1). Novel aspects of the present disclosureinclude, but are not limited to, the following specific variant peptidesbased on AM:

AM(37-52)NH₂ [Q50F]: (SEQ ID NO: 5) DKDNVAPRSKISPFGY;AM(37-52)NH₂ [Q50W]: (SEQ ID NO: 6) DKDNVAPRSKISPWGY;AM(37-52)NH₂ [K46L]: (SEQ ID NO: 7) DKDNVAPRSLISPQGY;AM(37-52)NH₂ [K46M]: (SEQ ID NO: 8) DKDNVAPRSMISPQGY;AM(37-52)NH₂ [K46W]: (SEQ ID NO: 9) DKDNVAPRSWISPQGY;AM(37-52)NH₂ [D39K]: (SEQ ID NO: 10) DKKNVAPRSKISPQGY;AM(37-52)NH₂ [S45W, Q50W]: (SEQ ID NO: 11) DKDNVAPRWKISPWGY;AM(37-52)NH₂ [K46L, Q50W]: (SEQ ID NO: 12) DKDNVAPRSLISPWGY;AM(37-52)NH₂ [D39W, Q50W]: (SEQ ID NO: 13) DKWNVAPRSKISPWGY;AM(37-52)NH₂ [D39K, Q50F]: (SEQ ID NO: 14) DKKNVAPRSKISPFGY;AM(37-52)NH₂ [D39K, Q50W]: (SEQ ID NO: 15) DKKNVAPRSKISPWGY;AM(37-52)NH₂ [K46L, Q50W, Y52F]: (SEQ ID NO: 16) DKDNVAPRSLISPWGF;AM(37-52)NH₂ [D39F, K46L, Q50W]: (SEQ ID NO: 17) DKFNVAPRSLISPWGY;AM(37-52)NH₂ [S45R, K46W, Q50W]: (SEQ ID NO: 18) DKDNVAPRRWISPWGY;AM(37-52)NH₂ [S45R, K46L, Q50W]: (SEQ ID NO: 19) DKDNVAPRRLISPWGY;AM(37-52)NH₂ [D39F, K46L, Q50W, Y52F]: (SEQ ID NO: 20) DKFNVAPRSLISPWGF;AM(37-52)NH₂ [S45W, K46W, Q50W, Y52F]: (SEQ ID NO: 21) DKDNVAPRWWISPWGF;AM(37-52)NH₂ [S45W, K46L, Q50W, Y52F]: (SEQ ID NO: 22) DKDNVAPRWLISPWGF;AM(37-52)NH₂ [S45W, K46M, Q50W, Y52F]: (SEQ ID NO: 23) DKDNVAPRWMISPWGF;AM(37-52)NH₂ [S45R, K46W, S48G, Q50W]: (SEQ ID NO: 24) DKDNVAPRRWIGPWGY;AM(37-52)NH₂ [S45R, K46L, S48G, Q50W]: (SEQ ID NO: 25) DKDNVAPRRLIGPWGY;AM(37-52)NH₂ [D37V, S45W, K46L, Q50W, Y52F]: (SEQ ID NO: 26)VKDNVAPRWLISPWGF; AM(37-52)NH₂ [S45W, K46L, S48G, Q50W, Y52F]:(SEQ ID NO: 27) DKDNVAPRWLIGPWGF;AM(37-52)NH2 [S45W, K46L, I47M, Q50W, Y52F]: (SEQ ID NO: 28)DKDNVAPRWLMSPWGF; AM(37-52)NH2 [S45R, K46W, I47L, S48G, Q50W]:(SEQ ID NO: 29) DKDNVAPRRWLGPWGY; andAM(37-52)NH2 [D39K, S45R, K46L, S48G, Q50W]: (SEQ ID NO: 30)DKKNVAPRRLIGPWGY.

Wild type human Calcitonin gene-related peptide (α-CGRP) is a 37 aminoacid, C-terminally-amidated peptide having the sequence:

ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF (SEQ ID NO:31), with a disulfidebond between the two cysteines.

Wild type human Calcitonin gene-related peptide (β-CGRP) is a 37 aminoacid, C-terminally-amidated peptide having the sequence:

ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF (SEQ ID NO:32), with a disulfidebond between the two cysteines.

CGRP(8-37)NH₂ is a 30 amino acid fragment of SEQ ID NO:31 having thesequence:

(SEQ ID NO: 33) VTHRLAGLLSRSGGVVKNNFVPTNVGSKAF.

CGRP(27-37)NH₂ is an 11 amino acid C-terminal fragment of SEQ ID NO:31having the sequence: FVPTNVGSKAF (SEQ ID NO:34).

FVPTDVGPFAF (SEQ ID NO:35) is a sequence published in Rist et al. (J MedChem. (1998) 41(1):117-23).

Variant peptides having sequences SEQ ID NOS: 36-43 below are mutants ofSEQ ID NO:34 (amino acids 27-37 of SEQ ID NO:31). Novel aspects of thepresent disclosure include, but are not limited to, the followingspecific variant peptides based on wild-type human α-CGRP and β-CGRP:

CGRP(27-37)NH₂ [N31D, S34P, K35W]: (SEQ ID NO: 36) FVPTDVGPWAF;CGRP(27-37)NH₂ [N31D, S34P, K35W, F37Y]: (SEQ ID NO: 37) FVPTDVGPWAY;CGRP(27-37)NH₂ [N31D, S34P, K35F, A36S]: (SEQ ID NO: 38) FVPTDVGPFSF;CGRP(27-37)NH₂ [N31D, S34P, K35W, A36S]: (SEQ ID NO: 39) FVPTDVGPWSF;CGRP(27-37)NH₂ [N31D, S34P, K35W, A36S, F37Y]: (SEQ ID NO: 40)FVPTDVGPWSY; CGRP(27-37)NH₂ [N31D, V32T, S34P, K35W, A36G]:(SEQ ID NO: 41) FVPTDTGPWGF;CGRP(27-37)NH₂ [N31D, V32T, S34P, K35W, A36S]: (SEQ ID NO: 42)FVPTDTGPWSF; and CGRP(27-37)NH₂ [N31D, V32T, S34P, K35W, A36G, F37Y]:(SEQ ID NO: 43) FVPTDTGPWGY.

Variant peptides having SEQ ID NOS: 44-46 below are mutants of thefragment comprising amino acids 30-37 of SEQ ID NO:31. Novel aspects ofthe present disclosure include, but are not limited to, the followingspecific variant peptides based on wild-type human α-CGRP and β-CGRP:

CGRP(30-37)NH₂ [N31D, S34P, K35W]: (SEQ ID NO: 44) TDVGPWAF;CGRP(30-37)NH₂ [N31D, S34P, K35W, A36S]: (SEQ ID NO: 45) TDVGPWSF; andCGRP(30-37)NH₂ [N31D, S34P, K35W, A36S, F37Y]: (SEQ ID NO: 46) TDVGPWSY.

Wild-type human adrenomedullin-2 (AM2)/intermedin is a 40 amino acid,C-terminally-amidated peptide having the sequence:

VGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO:47), with adisulfide bond between the two cysteines.

AM2/intermedin (1-24) is a fragment comprising the 24 N-terminal aminoacids of AM2/intermedin and having the sequence:

VGCVLGTCQVQNLSHRLWQLMGPA (SEQ ID. NO:48).

Embodiments of the present disclosure also include, but are not limitedto, variants of entire AM (SEQ ID NO:1), variant fragments thereof,and/or chimeric peptides comprising such variant fragments. Non-limitingexamples of such variant fragments are represented by the structure ofFormula I (SEQ ID NO:49), which is based on amino acids 37-52 of SEQ IDNO:1, and the structure of Formula II (SEQ ID NO:50), which is based onamino acids 42-52 of SEQ ID NO:1.

X₁-K-X₃-N-V-A-P-R-X₉-X₁₀-X₁₁-X₁₂-P-X₁₄-G-X₁₆   (I)

wherein

-   -   X₁ is A, D, V, L, or I,    -   X₃ is D, F, M, Y, V, I, E, K, W, L, or R,    -   X₉ is S, W, Y, F, R, T, or L,    -   X₁₀ is K, G, L, M, Y, A, W, or F,    -   X₁₁ is I, L, or M,    -   X₁₂ is S, T, or G,    -   X₁₄ is Q, F, Y, W, or H, and    -   X₁₆ is Y or F, and wherein the Y or F is amidated.

The peptides having the structure of Formula I may optionally beextended at the N-terminus by all or any portion of amino acids 1-36 ofSEQ ID NO:1, all or any portion of amino acids 1-22 of SEQ ID NO:31, allor any portion of amino acids 1-22 of SEQ ID NO:32, or all or anyportion of amino acids 1-24 of SEQ ID NO:47; and with the proviso thatthe variant peptide does not include a sequence comprising positions37-52 of SEQ ID NO:1. Examples of such extended portions include, butare not limited to, amino acids 13-36 of SEQ ID NO:1 and amino acids1-22 of SEQ ID NOs:31 or 32.

A-P-R-X₄-X₅-X₆-X₇-P-X₉-G-X₁₁   (II)

wherein

-   -   X₄ is S, W, Y, F, R, T, or L,    -   X₅ is K, G, L, M, Y, A, W, or F,    -   X₆ is I, L, or M,    -   X₇ is S, T, or G,    -   X₉ is Q, F, Y, W, or H, and    -   X₁₁ is Y or F, wherein the Y or F is amidated.

The peptides having the structure of Formula II may optionally beextended at the N-terminus by all or any portion of amino acids 1-41 ofSEQ ID NO:1, all or any portion of amino acids 1-26 of SEQ ID NO:31, allor any portion of amino acids 1-26 of SEQ ID NO:32, or all or anyportion of amino acids 1-29 of SEQ ID NO:47; and with the proviso thatthe variant peptide does not include a sequence comprising positions42-52 of SEQ ID NO:1. Examples of such extended portions include, butare not limited to, amino acids 13-41 of SEQ ID NO:1 and amino acids1-26 of SEQ ID NOs:31 or 32.

Embodiments of the present disclosure also include, but are not limitedto, variants of entire α-CGRP (SEQ ID NO:31) and β-CGRP (SEQ ID NO:32),variant fragments thereof, and/or chimeric proteins comprising suchvariant fragments. Non-limiting examples of such variant fragments arerepresented by the structure of Formula III (SEQ ID NO:51), which isbased on amino acids 27-37 of SEQ ID NO:31, and the structure of FormulaIV (SEQ ID NO:52), which is based on amino acids 30-37 of SEQ ID NO:31:

F-V-P-T-X₅-X₆-G-X₈-X₉-X₁₀-X₁₁   (III)

wherein

-   -   X₅ is N, D, or W,    -   X₆ is V, T, or W,    -   X₈ is P or S,    -   X₉ is W, Y, or H,    -   X₁₀ is A, S, or G,    -   X₁₁ is F or Y, wherein F or Y is amidated.

The peptides having the structure of Formula III may optionally beextended at the N-terminus by all or any portion of amino acids 1-41 ofSEQ ID NO:1, all or any portion of amino acids 1-26 of SEQ ID NO:31, allor any portion of amino acids 1-26 of SEQ ID NO:32, or all or anyportion of amino acids 1-29 of SEQ ID NO:47. Examples of such extendedportions include, but are not limited to, amino acids 13-41 of SEQ IDNO:1 and amino acids 1-26 of SEQ ID NOs:31 or 32.

T-X₂-X₃-G-X₅-X₆-X₇-X₈   (IV)

wherein

-   -   X₂ is N, D, or W,    -   X₃ is V, T, or W,    -   X₅ is P or S,    -   X₆ is W, Y, or H,    -   X₇ is A, S, or G,    -   X₈ is F or Y, wherein the F or Y is amidated.

The peptides having the structure of Formula IV may optionally beextended at the N-terminus by all or any portion of amino acids 1-44 ofSEQ ID NO:1, all or any portion of amino acids 1-29 of SEQ ID NO:31, allor any portion of amino acids 1-29 of SEQ ID NO:32, or all or anyportion of amino acids 1-32 of SEQ ID NO:47. Examples of such extendedportions include, but are not limited to, amino acids 13-44 of SEQ IDNO:1 and amino acids 1-23 or 1-29 of SEQ ID NOs:31 or 32.

The amino acid sequence having a structure as represented by Formula Vbelow was used to create variant peptide positional libraries (see FIG.8) based on positions 37-52 of AM:

D-K-D-N-V-A-P-R-X₉-X₁₀-X₁₁-X₁₂-P-W-G-X₁₆   (V)

wherein

-   -   X₉ is A, R, N, D, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or        V,    -   X₁₀ is A, R, N, D, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or        V,    -   X₁₁ is A, R, N, D, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or        V,    -   X₁₂ is A, R, N, D, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or        V, and    -   X₁₆ is A, R, N, D, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or        V, wherein X₁₆ is amidated.

The amino acid sequence having a structure as represented by Formula VIbelow was used to create variant peptide positional libraries (see FIG.9) based on positions 27-37 of CGRP:

F-V-P-T-X₅-X₆-X₇-P-W-X₁₀-X₁₁   (VI)

wherein

-   -   X₅ is A, R, N, D, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or        V,    -   X₆ is A, R, N, D, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or        V,    -   X₇ is A, R, N, D, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or        V,    -   X₁₀ is A, R, N, D, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or        V, and    -   X₁₁ is A, R, N, D, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or        V, wherein X₁₁ is amidated.

In at least certain embodiments, the present disclosure includes avariant peptide, optionally consisting of up to 70 amino acids, thatbinds to at least one receptor complex of the group consisting ofCLR:RAMP1, CLR:RAMP2, and CLR:RAMP3, the variant peptide having anaffinity K_(i) for the receptor complex of less than about 1 μM, thepeptide having a structure as represented by Formula I (SEQ ID NO:49):

X₁-K-X₃-N-V-A-P-R-X₉-X₁₀-X₁₁-X₁₂-P-X₁₄-G-X₁₆   (I)

wherein:

-   -   X₁ is A, D, V, L, or I,    -   X₃ is D, F, M, Y, V, I, E, K, W, L, or R,    -   X₉ is S, W, Y, F, R, T, or L,    -   X₁₀ is K, G, L, M, Y, A, W, or F,    -   X₁₁ is I, L, or M,    -   X₁₂ is S, T, or G,    -   X₁₄ is Q, F, Y, W, or H, and    -   X₁₆ is Y or F, wherein the Y or F is amidated;

wherein the variant peptide is optionally extended at the N-terminus byall or any portion of amino acids 1-36 of SEQ ID NO:1, by all or anyportion of amino acids 1-22 of SEQ ID NO:31, by all or any portion ofamino acids 1-22 of SEQ ID NO:32, or by all or any portion of aminoacids 1-24 of SEQ ID NO:47; and with the proviso that the variantpeptide does not include a contiguous sequence comprising the aminoacids in positions 37-52 of SEQ ID NO:1.

In certain embodiments, the variant peptide of Formula I has agonisticactivity for the at least one receptor complex. In certain otherembodiments, the variant peptide of Formula I has antagonistic activityfor the at least one receptor complex. The variant peptide may be boundto a carrier molecule directly or indirectly via a linker molecule.

In at least certain embodiments, the present disclosure includes avariant peptide, optionally consisting of up to 70 amino acids, thatbinds to at least one receptor complex of the group consisting ofCLR:RAMP1, CLR:RAMP2, and CLR:RAMP3, the variant peptide having anaffinity K_(i) for the receptor complex of less than about 1 μM, thepeptide having a structure as represented by Formula II (SEQ ID NO:50):

A-P-R-X₄-X₅-X₆-X₇-P-X₉-G-X₁₁   (II)

wherein:

-   -   X₄ is S, W, Y, F, R, T, or L,    -   X₅ is K, G, L, M, Y, A, W, or F,    -   X₆ is I, L, or M,    -   X₇ is S, T, or G,    -   X₉ is Q, F, Y, W, or H, and    -   X₁₁ is Y or F, wherein the Y or F is amidated;

wherein the peptide is optionally extended at the N-terminus by all orany portion of amino acids 1-41 of SEQ ID NO:1, by all or any portion ofamino acids 1-26 of SEQ ID NO:31, by all or any portion of amino acids1-26 of SEQ ID NO:32, or by all or any portion of amino acids 1-29 ofSEQ ID NO:47; and with the proviso that the variant peptide does notinclude a contiguous sequence comprising the amino acids in positions42-52 of SEQ ID NO:1.

In certain embodiments, the variant peptide of Formula II has agonisticactivity for the at least one receptor complex. In certain otherembodiments, the variant peptide of Formula II has antagonistic activityfor the at least one receptor complex. The variant peptide may be boundto a carrier molecule directly or indirectly via a linker molecule.

In at least certain embodiments, the present disclosure includes avariant peptide, optionally consisting of up to 70 amino acids, thatbinds to at least one receptor complex of the group consisting ofCLR:RAMP1, CLR:RAMP2, and CLR:RAMP3, the variant peptide having anaffinity K_(i) for the receptor complex of less than about 1 μM, thepeptide having a structure as represented by Formula III (SEQ ID NO:51):

F-V-P-T-X₅-X₆-G-X₈-X₉-X₁₀-X₁₁   (III)

wherein:

-   -   X₅ is N, D, or W,    -   X₆ is V, T, or W,    -   X₈ is P or S,    -   X₉ is W, Y, or H,    -   X₁₀ is A, S, or G,    -   X₁₁ is F or Y, wherein the F or Y is amidated;

wherein the peptide is optionally extended at the N-terminus by all orany portion of amino acids 1-41 of SEQ ID NO:1, by all or any portion ofamino acids 1-26 of SEQ ID NO:31, by all or any portion of amino acids1-26 of SEQ ID NO:32, or by all or any portion of amino acids 1-29 ofSEQ ID NO:47.

In certain embodiments, the variant peptide of Formula III has agonisticactivity for the at least one receptor complex. In certain otherembodiments, the variant peptide of Formula III has antagonisticactivity for the at least one receptor complex. The variant peptide maybe bound to a carrier molecule directly or indirectly via a linkermolecule.

In at least certain embodiments, the present disclosure includes avariant peptide, optionally consisting of up to 70 amino acids, thatbinds to at least one receptor complex of the group consisting ofCLR:RAMP1, CLR:RAMP2, and CLR:RAMP3, the variant peptide having anaffinity K_(i) for the receptor complex of less than about 1 μM, thepeptide having a structure of Formula IV (SEQ ID NO:52):

T-X₂-X₃-G-X₅-X₆-X₇-X₈   (IV)

wherein:

-   -   X₂ is N, D, or W,    -   X₃ is V, T, or W,    -   X₅ is P or S,    -   X₆ is W, Y, or H,    -   X₇ is A, S, or G,    -   X₈ is F or Y, wherein the F or Y is amidated;

wherein the peptide is optionally extended at the N-terminus by all orany portion of amino acids 1-44 of SEQ ID NO:1, by all or any portion ofamino acids 1-29 of SEQ ID NO:31, by all or any portion of amino acids1-29 of SEQ ID NO:32, or by all or any portion of amino acids 1-32 ofSEQ ID NO:47.

In certain embodiments, the variant peptide of Formula IV has agonisticactivity for the at least one receptor complex. In certain otherembodiments, the variant peptide of Formula IV has antagonistic activityfor the at least one receptor complex. The variant peptide may be boundto a carrier molecule, directly, or indirectly via a linker molecule.

As noted above, in certain embodiments, the variant peptides of thepresent disclosure (e.g., peptides comprising sequences constructedaccording to Formulas I-IV) can be linked, conjugated, complexed, bound,coupled, or otherwise attached to a carrier molecule, such as a proteinor polymeric material such as polyethylene glycol (PEG) via PEGylation.For example, the variant peptides may be conjugated or otherwiseattached to a suitable carrier molecule such as, but not limited to,keyhole limpet haemocyanin (KLH), ovalbumin, bovine serum albumin (BSA),or human serum albumin (HSA). Other examples of carrier proteins whichmay be used include, but are not limited to, those disclosed in USPublished Patent Applications 2013/0072881, 2013/0209503, and2013/0337006, the disclosures of which are expressly incorporated hereinby reference. The variant peptide may be bound directly to the carriermolecule or linked via a linker compound or linker peptide, for exampleas described in the US Published Patent Applications 2013/0072881,2013/0209503, and 2013/0337006.

As noted above, the variant peptides of the present disclosure (e.g.,peptides comprising sequences constructed according to Formulas I-IV)can be used to treat a number of conditions and diseases involving theCGRP and AM receptor complexes CLR:RAMP 1, CLR:RAMP2, and CLR:RAMP3.Examples of conditions and diseases that can be treated in a subject bythe administration of a variant peptide of the present disclosure whichhas AM receptor antagonistic activity include, but are not limited to:Prostate cancer, Glioblastoma, Lewis lung carcinoma, Cervical cancer,Melanoma, Breast cancer, Colon cancer, Ovarian cancer, and Sepsis.Examples of conditions and diseases that can be treated in a subject bythe administration of a variant peptide of the present disclosure whichhas AM receptor agonistic activity include, but are not limited to:Ulcerative colitis, Crohn's disease, Inflammatory bowel disease,Myocardial infarction, Heart failure, Atherosclerotic vascular disease,Tissue or organ ischemia, Arteriosclerosis obliterans, Buerger'sdisease, Ischemic brain injury, Pulmonary hypertension, Sepsis,Lymphedema (primary and secondary), Promotion of embryo implantationduring in vitro fertilization, and Preeclampsia. Examples of conditionsand diseases that can be treated in a subject by the administration of avariant peptide of the present disclosure which has CGRP receptorantagonistic activity include, but are not limited to: Migraineheadache, Hyperalgesia, Menopausal hot flashes, Arthritis (bothosteoarthritis and rheumatoid arthritis), and Sepsis. Examples ofconditions and diseases that can be treated in a subject by theadministration of a variant peptide of the present disclosure which hasCGRP receptor agonistic activity include, but are not limited to:Pulmonary hypertension, Heart failure, Atherosclerosis, sepsis,Myocardial ischemia, gut ischemia, liver ischemia, kidney ischemia, andbrain ischemia.

The embodiments of the present disclosure will be more readilyunderstood by reference to the following examples and embodiments, whichare included merely for purposes of illustration of certain aspects andembodiments of the inventive concepts, and are not intended to belimiting. The following detailed examples and methods describe how tomake and use the various mutant peptides of the present disclosure andare to be construed, as noted above, only as illustrative, and notlimitations of the disclosure in any way whatsoever. Those skilled inthe art will promptly recognize appropriate variations from thematerials and procedures described herein.

EXAMPLE

Methods

Protein production and characterization and peptides. Plasmidconstruction, mutagenesis, protein expression, purification, and theAlphaScreen® peptide binding assay (Perkin-Elmer, Waltham, Mass.) wereperformed as previously described with minor modifications to theAlphaScreen® assay (Hill and Pioszak, Protein expression andpurification (2013) 88:107-113; and Moad and Pioszak, Protein Sci.(2013) 22:1775-85). Synthetic peptides were from RS Synthesis(Louisville, Ky.), Bachem (Bubendorf, Switzerland), or were synthesizedin-house. Bacterial pETDuet1 expression plasmids encoding tetheredMBP-hRAMP1.24-111-(Gly-Ser-Ala)₃-hCLR.29-144-H₆ andMBP-hRAMP2.55-140-(Gly-Ser-Ala)₃-hCLR.29-144-H₆ (amino acid numbersindicated) fusion proteins co-expressed with DsbC were constructed usingthe Gibson Assembly cloning method with Gibson Assembly master mix (NewEngland Biolabs, Ipswich, Mass.). Plasmids for expression of taggedfull-length CLR and RAMP1 or RAMP2 receptors in mammalian cells werepreviously described (Barwell et al., Peptides (2010) 31:170-176;Watkins et al., Br J Pharmacol (2014) 171:772-788). Site directedmutagenesis was performed with the QuikChange II kit (AgilentTechnologies, Santa Clara, Calif.) or using the Gibson Assembly method.All constructs were verified by automated DNA sequencing.

The tethered ECD fusion proteins were expressed in E. coli, purified,and characterized for peptide binding with an AlphaScreen® peptidebinding assay (Perkin-Elmer, Waltham, Mass.) as previously described(Moad and Pioszak, Protein Sci. (2013) 22:1775-85), except that theAlphaScreen® competition assays also included 0.3% (v/v) Triton X-100 inthe reaction buffer. Triton X-100 minimized, but did not completelyprevent, apparent aggregation of the CGRP(27-37)NH₂[N31D,S34P,K35F]peptide (SEQ ID NO:35) that was surprisingly observed only with theMBP-hRAMP1.24-111-(Gly-Ser-Ala)₃-hCLR.29-144-H₆ protein and not withMBP-hRAMP1.24-111-(Gly-Ser)₅-hCLR.29-144-H₆ or MBP-hRAMP2.55-140[L106R]-(Gly-Ser-Ala)₃-hCLR.29-144-H₆ proteins (data not shown). Theapparent aggregation of the CGRP analog prevented it from fullycompeting the binding signal to background levels, and hence thereported IC₅₀ values for the CGRP analog peptides binding to the CGRPreceptor crystallization construct are likely a bit higher than the truevalues. Competitor peptide concentrations higher than 200 μM wereavoided because some of the peptides began to exhibit non-specificinhibition at concentrations >200 μM as assessed in control reactions inwhich the donor and acceptor beads were brought together by aBiotin-(Gly)₆-(His)₆ peptide. The binding experiments were conducted atleast three times with each independent experiment performed withduplicate samples. pIC₅₀ values are stated as the mean of the replicateindependent experiments ±S.E.M. Although slight variation in pIC₅₀values for a given peptide in assays conducted on different days wasoccasionally observed, the rank order of IC₅₀ values for the variouspeptides and the magnitude of their differences were very reproducible.

Fluorescence polarization peptide binding assay. The ability of theindicated peptides to displace a FITC-Ahx-AM(37-52)NH₂[S45W/Q50W] probe(7 nM) from purified MBP-RAMP1 ECD-(GS)₅-CLR ECD-H₆ fusion protein (15nM) or from purified MBP-RAMP2 ECD [L106R]-(GS)₅-CLR ECD-H₆ fusionprotein (110 nM) was assessed by fluorescence polarization (anisotropy).The reactions (50 μL) were incubated for 2 hours at room temperature toreach equilibrium, and the anisotropy was measured in black half-area96-well plates using a BMG LABTECH's POLARstar® Omega plate reader(Ortenberg, Germany). The reaction buffer was 50 mM HEPES, pH 7.5, 150mM NaCl, 0.5 mg/ml fatty acid-free BSA, 0.5 mM EDTA, 0.5 mM maltose.K_(I) values were calculated using nonlinear regression fitting of thecompetition curves to the exact analytical equations of Roehrl et al.(Biochemistry (2004) 43:16056-66) using GraphPad Prism 5.0 (GraphPadSoftware, San Diego Calif.).

Peptides. Custom synthetic peptides or peptide libraries for bindingstudies and crystallization were obtained from RS Synthesis (Louisville,Ky.) except the CGRP(27-37)NH₂[N31D,S34P,K35F] peptide used forcrystallization, which was assembled by Fmoc SPPS on Rink amidepolystyrene resin using a Tribute synthesizer (Protein Technologies,Tucson, Ariz.) with 20% (v/v) piperidine in DMF as Fmoc deblockingreagent (2×5 mins) and HATU/DIPEA (20 mins) as coupling reagents. Thepeptide was cleaved from the resin with concomitant removal of sidechain protecting groups with 95% TFA/2.5% TIPS/2.5% water (v/v/v) for 2hours and recovered by precipitation into cold diethyl ether andisolated by centrifugation (221 mg). Purification of a portion (110 mg)by RP-HPLC on a C18 column (Waters Xterra, 19×300 mm) afforded the titlecompound (27.4 mg, >95% purity by HPLC), observed mass (ESI+)(M+H)¹⁺=1195.0, calculated mass 1196.4. For cell-based assays humanAM(1-52) was from Bachem (Bubendorf, Switzerland) and human αCGRP(1-37)was synthesized in-house (PWH) or was from Bachem.

Crystallization, structure solution, and homology modeling. The tetheredMBP-RAMP1 ECD-CLR ECD and MBP-RAMP2 ECD [L106R]-CLR ECD proteins werecomplexed with CGRP(27-37)NH₂[N31D,S34P,K35F] (SEQ ID NO:35) orAM(25-52)NH₂ (amino acids 25-52 of SEQ ID NO:1) and crystallized with areservoir solution of 22% PEG3350, 8% Tacsimate, pH 6.0 for the CGRPreceptor complex or 19% PEG3350, 0.1 M Tris-HCl, pH 8.3, 225 mM sodiumacetate, and 20% ethylene glycol for the AM₁ receptor complex.Diffraction data collected at the APS synchrotron (Argonne, Ill.) wereprocessed with HKL2000 (Otwinowski and Minor, (1997) Processing of X-raydiffraction data collected in oscillation mode. In Methods inEnzymology, C. W. J. Carter, and R. M. Sweet, eds. (New York: AcademicPress), pp. 307-326.) and the CCP4 suite (Winn et al., Acta CrystallogrD Biol Crystallogr (2011) 67:235-242). The structures were solved bymolecular replacement with Phaser (McCoy et al., J Appl Crystallogr(2007) 40:658-674), rebuilt with COOT (Emsley et al., Acta Crystallogr DBiol Crystallogr (2010) 66:486-501), and refined with REFMACS (Murshudovet al., Acta Crystallogr D Biol Crystallogr (1997) 53:240-255).

The tethered MBP-RAMP1 ECD-CLR ECD and MBP-RAMP2 ECD [L106R]-CLR ECDfusion proteins were incubated for 1 hour on ice in the presence ofCGRP(27-37)NH₂[N31D,S34P,K35F] or AM(25-52)NH₂ (1:1.3 protein:peptidemolar ratio), respectively, in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mMEDTA, 1mM maltose and spin concentrated to 30 mg/ml for crystallization.Crystals were grown by the hanging drop vapor diffusion method at 20° C.with a reservoir solution of 22% PEG3350, 8% Tacsimate (HamptonResearch), pH 6.0 for the CGRP receptor complex or 19% PEG3350, 0.1 MTris-HCl, pH 8.3, 225 mM sodium acetate, and 20% ethylene glycol for theAM₁ receptor complex. Microseeding was used to obtain the best CGRPreceptor complex crystals for data collection. CGRP receptor complexcrystals were cryoprotected by dialysis to mother liquor solutioncontaining 12% PEG400; AM₁ receptor complex crystals were suitablycryoprotected in their growth condition. Crystals were flash frozen inliquid nitrogen and diffraction data were collected remotely at beamline21-ID-G (λ=0.97857 Å) of the Advanced Photon Source (Argonne, Ill). Datafrom single crystals were indexed, integrated, and scaled with HKL2000v. 705b (Otwinowski and Minor, (1997). Processing of X-ray diffractiondata collected in oscillation mode. In Methods in Enzymology, C. W. J.Carter, and R. M. Sweet, eds. (New York: Academic Press), pp. 307-326)and further processed/analyzed with the CCP4 suite v.6.4.0 (Winn et al.,Acta Crystallogr D Biol Crystallogr (2011) 67:235-242) in preparationfor molecular replacement (MR). The structures were solved with Phaserv. 2.5.6 (McCoy et al., J Appl Crystallogr (2007) 40:658-674) using anMBP search model with maltose removed (PDB 3C4M) followed by ligand-freeCLR:RAMP1 ECD (PDB 3N7S) or CLR:RAMP2 ECD heterodimer search models (PDB3AQF). The MR solutions were rigid body refined with REFMACS v. 5.8.0073(Murshudov et al., Acta Crystallogr D Biol Crystallogr (1997)53:240-255) treating MBP, CLR, and RAMP1 or RAMP2 as separate rigidbodies. At this stage 2mF_(o)-DF_(c) and mF_(o)-DF_(c) electron densitymaps clearly showed bound maltose and CGRPmut or AM peptide. The modelswere completed by iterative rounds of manual rebuilding in COOT (Emsleyet al., Acta Crystallogr D Biol Crystallogr (2010) 66:486-501) and TLSand restrained refinement with REFMACS. NCS restraints were applied tothe three molecules in the ASU of the CGRP receptor complex structurewith the restraints relaxed for areas where the molecules differed.Structure analysis used PyMol (Schrodinger) and programs in the CCP4suite and figures were prepared with PyMol. Structural superpositionswere performed with the PyMol align command (for Ca atoms) utilizingoutlier rejection.

Homology Modeling. Homology models of RAMP3 were generated usingModeller 9v12 (Sali and Blundell, J Mol Biol (1993) 234:779-815).CGRPmut-bound CLR:RAMP1 and AM-bound CLR:RAMP2 template structures wereused either singularly or in combination to generate the models. 6000models were generated, refined using Rosetta 3.5 (Rohl et al., MethodsEnzymol (2004) 383:66-93) and ranked using the OPUS_PSP scoring function(Lu et al., J Mol Biol (2008) 376, 288-301). The 600 best scoringstructures were then clustered into 0.1 nm bins using the g clusterfunction as implemented in Gromacs (Pronk et al., Bioinformatics (2013)29:845-854). The best scoring structure from the largest, best scoringcluster was then selected.

Cell-based assays. Transfection of COS-7 cells, cAMP assay, ELISA forcell surface expression, and data analysis were as previously described(Barwell et al., Peptides (2010) 31:170-176; Watkins et al., Br JPharmacol (2014) 171:772-788).

Accession Numbers: Coordinates and structure factors were deposited inthe RCSB Protein Data Bank with codes 4RWF and 4RWG for the AM:CLR:RAMP2and CGRPmut:CLR:RAMP1 complexes, respectively.

Results

FIG. 1 depicts in (A) binding of a CGRP C-terminal peptide,CGRP(8-37)NH₂, and two variant AM C-terminal peptides(AM(37-52)NH₂[Q50W] and AM(37-52)NH₂[S45W,Q50W]) to purified CGRPreceptor extracellular domain complex (CLR:RAMP1). FIG. 1 depicts in (B)binding of an AM C-terminal peptide, AM(37-52)NH₂, and the two variantAM C-terminal peptides to purified AM₁ receptor extracellular domaincomplex (CLR:RAMP2). The ability of the indicated peptides to displace aBiotin-CGRP probe (50 nM) from purified MBP-RAMP1 ECD-(GS)₅-CTR ECD-H₆fusion protein (5 nM) or a Biotin-AM probe (25 nM) from purifiedMBP-RAMP2 ECD [L106R]-(GS)₅-CTR ECD-H₆ fusion protein (5 nM) wasassessed by AlphaLISA® luminescent peptide binding assay (Perkin-Elmer,Waltham, Mass.). The probes were as described in Moad and Pioszak(Protein Sci. (2013) 22:1775-85). The reactions were incubated for 5hours at room temperature to reach equilibrium, and the luminescence wasmeasured in white 384-well plates using a BMG LABTECH's POLARstar® Omegaplate reader (Ortenberg, Germany). The reaction buffer was 50 mM MOPS,pH 7.4, 150 mM NaCl, 7 mg/ml fatty acid-free BSA, and thestreptavidin-coated donor beads and MBP-antibody coated acceptor beadswere at 15 μg/ml each. IC₅₀ values were calculated using nonlinearregression fitting of the competition curves to a three-parameter,one-site competitive binding equation in GraphPad Prism 5.0 (GraphPadSoftware, San Diego Calif.).

FIG. 2 depicts binding of (A) AM-based peptide AM(37-52)NH₂; (B) variantpeptides AM(37-52)NH₂[S45W, Q50W], AM(37-52)NH₂[Q50F], andAM(37-52)NH₂[Q50W]; (C) variant peptides AM(37-52)NH₂[D39K],AM(37-52)NH₂[K46L, Q50W], and AM(37-52)NH₂[K46L, Q50W, Y52F]; (D)variant peptides AM(37-52)NH₂[D39F, K46L, Q50W], AM(37-52)NH₂[D39F,K46L, Q50W, Y52F], and AM(37-52)NH₂[S45W, K46L, Q50W, Y52F]; (E) variantpeptides AM(37-52)NH₂[S45W, K46M, Q50W, Y52F], and AM(37-52)NH₂[S45W,K46W, Q50W, Y52F]; (F) variant peptides AM(37-52)NH₂[S45T, K46W, Q50W],AM(37-52)NH₂[S45T, K46L, Q50W], and AM(37-52)NH₂[S45W, K46L, S48G, Q50W,Y52F]; (G) variant peptide AM(37-52)NH₂[S45W, K46L, I47M, Q50W, Y52F];(H) variant peptides AM(37-52)NH₂[S45R, K46W, Q50W], andAM(37-52)NH₂[S45R, K46L, Q50W]; (I) variant peptides AM(37-52)NH₂[S45R,K46W, S48G, Q50W], AM(37-52)NH₂[S45R, K46L, S48G, Q50W], andAM(37-52)NH₂[S45R, K46W, I47L, S48G, Q50W]; and (J) variant peptidesAM(37-52)NH₂[D39W, Q50W], and AM(37-52)NH₂[D37V, S45W, K46L, Q50W,Y52F], to the purified CGRP receptor extracellular domain complex(CLR:RAMP1). The ability of the indicated peptides to displace aFITC-Ahx-AM(37-52)NH₂ [S45W, Q50W] probe (7 nM) from purified MBP-RAMP1ECD-(GS)₅-CLR ECD-H₆ fusion protein (15 nM) was assessed by fluorescencepolarization (anisotropy). The reactions were incubated for 2 hours atroom temperature to reach equilibrium, and the anisotropy was measuredin black half-area 96-well plates using a BMG LABTECH's POLARstar® Omegaplate reader (Ortenberg, Germany). The reaction buffer was 50 mM HEPES,pH 7.5, 150 mM NaCl, 0.5 mg/ml fatty acid-free BSA, 0.5 mM EDTA, 0.5 mMmaltose. K_(I) values were calculated using nonlinear regression fittingof the competition curves to the exact analytical equations of Roehrl etal. (Biochemistry (2004) 43:16056-66) using GraphPad Prism 5.0 (GraphPadSoftware, San Diego Calif.).

FIG. 3 depicts binding of (A) AM-based peptide AM(37-52)NH₂, and variantpeptide AM(37-52)NH₂[S45W, Q50W]; (B) AM-based peptide AM(37-52)NH₂,variant peptide AM(37-52)NH₂[Q50F], and variant peptideAM(37-52)NH₂[Q50W]; (C) variant peptides AM(37-52)NH₂[K46L],AM(37-52)NH₂[K46M], and AM(37-52)NH₂[D39F]; (D) variant peptidesAM(37-52)NH₂[D39F, K46L, Q50W], AM(37-52)NH₂[D39F, K46L, Q50W, Y52F],and AM(37-52)NH₂[S45W, K46L, Q50W, Y52F]; (E) variant peptidesAM(37-52)NH₂[[D39K], AM(37-52)NH₂[K46L, Q50W], and AM(37-52)NH₂[K46L,Q50W, Y52F]; (F) variant peptides AM(37-52)NH₂[S45W, K46M, Q50W, Y52F],and AM(37-52)NH₂[S45W, K46W, Q50W, Y52F]; (G) variant peptidesAM(37-52)NH₂[S45T, K46W, Q50W], AM(37-52)NH₂[S45T, K46L, Q50W], andAM(37-52)NH₂[S45W, K46L, S48G, Q50W, Y52F]; (H) variant peptideAM(37-52)NH₂[S45W, K46L, I47M, Q50W, Y52F]; (I) variant peptidesAM(37-52)NH₂[S45R, K46W, Q50W], and AM(37-52)NH₂[S45R, K46L, Q50W]; (J)variant peptides AM(37-52)NH₂[S45R, K46W, S48G, Q50W],AM(37-52)NH₂[S45R, K46L, S48G, Q50W], and AM(37-52)NH₂[S45R, K46W, I47L,S48G, Q50W]; and (K) variant peptides AM(37-52)NH₂[D39W, Q50W], andAM(37-52)NH₂[D37V, S45W, K46L, Q50W, Y52F], to the purified AM₁ receptorextracellular domain complex. Single representative experimentsconducted with duplicate samples are shown. Error bars are S.E.M. Theability of the indicated peptides to displace aFITC-Ahx-AM(37-52)NH₂[S45W, Q50W] probe (7 nM) from purified MBP-RAMP2ECD [L106R]-(GS)₅-CLR ECD-H₆ fusion protein (110 nM) was assessed byfluorescence polarization (anisotropy). The reactions were incubated for2 hours at room temperature to reach equilibrium, and the anisotropy wasmeasured in black half-area 96-well plates using a BMG LABTECH'sPOLARstar® Omega plate reader (Ortenberg, Germany). The reaction bufferwas 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mg/ml fatty acid-free BSA, 0.5mM EDTA, 0.5 mM maltose. K_(I) values were calculated using nonlinearregression fitting of the competition curves to the exact analyticalequations of Roehrl et al. (Biochemistry (2004) 43:16056-66) usingGraphPad Prism 5.0 (GraphPad Software, San Diego Calif.).

FIG. 4 depicts binding of (A) CGRP-based peptide CGRP[8-37]NH₂; (B)variant peptides CGRP(27-37)NH₂[N31D, S34P, K35F], CGRP(27-37)NH₂[N31D,S34P, K35W], and CGRP(27-37)NH₂[N31D, S34P, K35F, A36S]; (C) variantpeptides CGRP(27-37)NH₂[N31D, S34P, K35W, A36S], andCGRP(27-37)NH₂[N31D, S34P, K35W, A36S, F37Y]; (D) variant peptidesCGRP(27-37)NH₂[N31D, V32T, S34P, K35W, A36G], and CGRP(27-37)NH₂[N31D,V32T, S34P, K35W, A36G, F37Y]; and (E) variant peptideCGRP(27-37)NH₂[N31D, V32T, S34P, K35W, A36S], to purified CGRP receptorextracellular domain complex. Single representative experimentsconducted with duplicate samples are shown. Error bars are S.E.M. Theability of the indicated peptides to displace aFITC-Ahx-AM(37-52)NH₂[S45W, Q50W] probe (7 nM) from purified MBP-RAMP1ECD-(GS)₅-CLR ECD-H₆ fusion protein (15 nM) was assessed by fluorescencepolarization (anisotropy). The reactions were incubated for 2 hours atroom temperature to reach equilibrium, and the anisotropy was measuredin black half-area 96-well plates using a BMG LABTECH's POLARstar® Omegaplate reader (Ortenberg, Germany). The reaction buffer was 50 mM HEPES,pH 7.5, 150 mM NaCl, 0.5 mg/ml fatty acid-free BSA, 0.5 mM EDTA, 0.5 mMmaltose. K_(I) values were calculated using nonlinear regression fittingof the competition curves to the exact analytical equations of Roehrl etal. (Biochemistry (2004) 43:16056-66) using GraphPad Prism 5.0 (GraphPadSoftware, San Diego Calif.).

FIG. 5 depicts binding of (A) CGRP-based peptide CGRP(11-37)NH₂; (B)variant peptides CGRP(27-37)NH₂[N31D, S34P, K35F], CGRP(27-37)NH₂[N31D,S34P, K35W], and CGRP(27-37)NH₂[N31D, S34P, K35F, A36S]; (C) variantpeptides CGRP(27-37)NH₂[N31D, S34P, K35W, A36S], andCGRP(27-37)NH₂[N31D, S34P, K35W, A36S, F37Y]; (D) variant peptidesCGRP(27-37)NH₂[N31D, V32T, S34P, K35W, A36G], and CGRP(27-37)NH₂[N31D,V32T, S34P, K35W, A36G, F37Y]; and (E) variant peptideCGRP(27-37)NH₂[N31D, V32T, S34P, K35W, A36S], to the purified AM₁receptor extracellular domain complex. Single representative experimentsconducted with duplicate samples are shown. Error bars are S.E.M. Theability of the indicated peptides to displace aFITC-Ahx-AM(37-52)NH₂[S45W, Q50W] probe (7 nM) from purified MBP-RAMP2ECD [L106R]-(GS)₅-CLR ECD-H₆ fusion protein (110 nM) was assessed byfluorescence polarization (anisotropy). The reactions were incubated for2 hours at room temperature to reach equilibrium, and the anisotropy wasmeasured in black half-area 96-well plates using a BMG LABTECH'sPOLARstar® Omega plate reader (Ortenberg, Germany). The reaction bufferwas 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mg/ml fatty acid-free BSA, 0.5mM EDTA, 0.5 mM maltose. K_(I) values were calculated using nonlinearregression fitting of the competition curves to the exact analyticalequations of Roehrl et al. (Biochemistry (2004) 43:16056-66) usingGraphPad Prism 5.0 (GraphPad Software, San Diego Calif.).

FIG. 6 depicts binding of CGRP variant peptide CGRP(27-37)NH₂ [N31D,S34P, K35W, A36S] and AM variant peptide AM(37-52)NH₂ [S45W, K46L, QSOW,Y52F], to purified AMY₁ receptor extracellular domain complex. A singlerepresentative experiment conducted with duplicate samples is shown.Error bars are S.E.M. The ability of the indicated peptides to displacea Biotin-AC413 probe (100 nM) from purified MBP-RAMP1 ECD-(GS)₅-CTRECD-H₆ fusion protein (200 nM) was assessed by AlphaLISA® luminescentpeptide binding assay (Perkin-Elmer, Waltham, Mass.) as described in Leeet al. (J Biol. Chem (2016) 291:8686-8700). The reactions were incubatedfor 5 hours at room temperature to reach equilibrium, and theluminescence was measured in white 384-well plates using a BMG LABTECH'sPOLARstar® Omega plate reader (Ortenberg, Germany). The reaction bufferwas 50 mM MOPS, pH 7.4, 150 mM NaCl, 7 mg/ml fatty acid-free BSA and thestreptavidin-coated donor beads and MBP-antibody coated acceptor beadswere at 15 μg/ml each. IC₅₀ values were calculated using nonlinearregression fitting of the competition curves to a three-parameter,one-site competitive binding equation in GraphPad Prism 5.0 (GraphPadSoftware, San Diego Calif.). The IC₅₀ values for CGRP (27-37)NH₂[N31D,S34P, K35W, A36S] and AM (37-52)NH₂[S45W, K46L, Q50W, Y52F] were 260 nMand 16 μM, respectively.

FIG. 7 depicts antagonism of cAMP signaling at full-length (A) CGRP(RAMP1:CLR) and (B) AM₁ (RAMP2:CLR) receptor complexes transientlyexpressed in COS-7 cells by the variant peptide AM(37-52)NH₂[S45W, K46L,Q50W, Y52F]. A single representative experiment conducted with duplicatesamples is shown. Error bars are S.E.M. The experiments were performedas described in Lee et al. (J Biol. Chem (2016) 291:8686-8700). (A)depicts antagonism of CGRP-activated cAMP signaling at the CGRPreceptor. The pA₂ value calculated by fitting the curves to aGaddum/Schild EC₅₀ shift equation with Hill and Schild slopesconstrained to 1 in GraphPad Prism 5.0 was 7.98. (B) depicts antagonismof AM-activated cAMP signaling at the AM₁ receptor.

FIG. 8 depicts binding of AM-based PS-SPCL library mixtures to purifiedCGRP (A, C, E) and AM₁ (B, D, F) receptor extracellular domaincomplexes. One of 5 positions of the 16 positions in variantAM(37-52)NH₂[Q50W] was optimized for each mixture. In (A, B) position 45was optimized, in (C, D) position 46 was optimized, in (E, F) position47 was optimized, in (G, H) position 48 was optimized, and in (I, J)position 52 was optimized. The ability of the indicated mixtures todisplace a FITC-Ahx-AM(37-52)NH₂ [S45W, Q50W] probe (7 nM) from purifiedMBP-RAMP1 ECD-(GS)₅-CLR ECD-H₆ fusion protein (15 nM) or MBP-RAMP2 ECD[L106R]-(GS)₅-CLR ECD-H₆ fusion protein (110 nM) was assessed byfluorescence polarization (anisotropy). The reactions were incubated for2 hours at room temperature to reach equilibrium, and the anisotropy wasmeasured in black half-area 96-well plates using a BMG LABTECH'sPOLARstar® Omega plate reader (Ortenberg, Germany). The reaction bufferwas 100 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mg/ml fatty acid-free BSA,0.5 mM EDTA, 0.5 mM maltose. The final library mixture concentrationswere 0.05 mg/ml for the CGRP receptor ECD complex and 0.5 mg/ml for theAM₁ receptor ECD complex.

FIG. 9 depicts binding of CGRP-based PS-SPCL library mixtures topurified CGRP receptor extracellular domain complexes. One of 5positions of the 11 positions in variant CGRP(27-37)NH₂[S34P, K35W] wasoptimized for each mixture. In (A) position 31 was optimized, in (B)position 32 was optimized, in (C) position 33 optimized, in (D) position36 optimized, and in (E) position 37 optimized. Single experimentsconducted with duplicate samples are shown. Error bars are S.E.M. (−)indicates no competitor control. The ability of the indicated mixturesto displace a FITC-Ahx-AM(37-52)NH₂ [S45W, Q50W] probe (7 nM) frompurified MBP-RAMP1 ECD-(GS)₅-CLR ECD-H₆ fusion protein (15 nM) wasassessed by fluorescence polarization (anisotropy). The reactions wereincubated for 2 hours at room temperature to reach equilibrium, and theanisotropy was measured in black half-area 96-well plates using a BMGLABTECH's POLARstar® Omega plate reader (Ortenberg, Germany). Thereaction buffer was 100 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mg/ml fattyacid-free BSA, 0.5 mM EDTA, 0.5 mM maltose. The final library mixtureconcentrations were 0.11 mg/ml. The peptide mixture for Q at position 37was problematic for unknown reasons and was therefore omitted.

Discussion

Tethered RAMP1 ECD- and RAMP2 ECD-CLR ECD fusion proteins wereengineered to enhance complex stability (Moad and Pioszak. Protein Sci.(2013) 22:1775-85). The fusions selectively bound their respectivepeptides with μM affinities. Structures of RAMP1-CLR ECD in complexbound with a high-affinity antagonist CGRP analog (“CGRPmut”—SEQ IDNO:35), and RAMP2-CLR ECD bound to an antagonist AM fragment, revealingthat the peptides occupy a shared binding site on CLR with largelyunstructured conformations characterized by a β-turn near theirC-terminus. AM contains a single α-helical turn that is absent inCGRPmut. These conformations contrast the continuous α-helices formed byother ECD-bound class B GPCR peptide ligands. Two key areas of the CLRpeptide-binding site are a hydrophobic patch and a pocket that isaugmented by the RAMP. Prior to the β-turn, the peptides contact thepatch, and after the β-turn, their C-terminal residue occupies thepocket. CGRPmut F37 makes hydrophobic contact with RAMP1 W84, and AM Y52forms a key hydrogen bond with RAMP2 E101. The structures and a RAMP3homology model (not shown) explain how these RAMP-peptide contactsaffect selectivity. RAMP2 E101 and RAMP3 E74 favor AM by H-bonding withY52. RAMP1 W74 precludes this bond. RAMP1 W84 enables contact with CGRPF37, whereas the smaller F111 at the equivalent RAMP2 position precludesthis contact, thereby disfavoring CGRP binding. RAMP3 W84 probablyexplains why CGRP is more potent at AM₂ than AM₁. RAMP-peptide contactclearly is important for ligand selectivity at CLR, but allostery mayalso play a role. Swapping the CGRP and AM C-terminal residues did notswitch their receptor selectivity, which suggested that RAMP-peptidecontact alone cannot explain selectivity. Subtle differences in CLRconformation in the two structures appear to be RAMP-dependent includingdifferent positions of CLR R119 and slightly different pocket shapes.

Development of AM and CGRP Variants with Enhanced Potency andSelectivity for the CGRP or AM Receptors.

Our recent crystal structures of the CGRPmut:RAMP1-CLR ECD andAM:RAMP2-CLR ECD complexes at resolutions of 2.5 and 1.8 Å,respectively, revealed the structural basis for peptide recognition andprovided insights into how RAMPs determine ligand selectivity. In ourcrystal structure of peptide-bound RAMP1-CLR ECD, a high-affinity CGRPanalog was used, because it yielded higher-quality crystals than CGRP.This analog, “CGRPmut” (SEQ ID NO:35), was developed by Rist et al. (JMed Chem. (1998) 41(1):117-23) using systematic mutagenesis in which theC-terminal antagonist fragment (27-37) was scanned with several aminoacids. The results revealed the basis for the enhanced affinity. TheN31D substitution does not appear to provide affinity enhancement, butit probably increases peptide solubility. The S34P substitution promotesβ-turn formation equivalent to AM P49, and K35F increases hydrophobiccontact to CLR loop4 (FIG. 1A). It was hypothesized that substitution ofAM Q50 (equivalent to CGRP K35) with F or W would similarly enhanceaffinity. Using the molecular graphics software Pymol (Schrodinger), insilico mutagenesis was performed to model substitutions at AM Q50 andother positions. The modeling suggested that Q50W would contact CLR S117and R119 in loop4 and that the addition of S45W in a double mutant wouldallow stacking of the Trp residues, which may stabilize the β-turn.Custom synthesized and HPLC-purified AM(37-52) antagonist peptidescontaining the Q50W and S45W/Q50W mutations were synthesized and testedfor binding to the CGRP and AM₁ receptor ECD complexes using anAlphaLISA® luminescent proximity assay (Perkin-Elmer, Waltham, Mass.)with purified tethered MBP-RAMP1 ECD-CLR ECD and MBP-RAMP2 ECD-CLR ECDfusion proteins as described. All data used purified MBP-RAMPECD-CLR/CTR ECD fusions; “MBP” is often omitted hereafter. Humanpeptides and receptors were used unless otherwise noted, and allpeptides were C-terminally amidated.

In competition assays, the AM(37-52)NH₂[Q50W] single mutant had˜100-fold increased affinity for RAMP2-CLR ECD and significantlyenhanced affinity for RAMP1-CLR ECD. The S45W/Q50W double mutant did notappear to further enhance binding to RAMP2-CLR ECD, but it enhancedbinding to RAMP1-CLR ECD such that it was non-selective with apparent nMaffinity. AlphaLISA®/AlphaScreen® can detect weak molecular interactions(μM K_(d)) using nM concentrations of binding partners in part due tomultivalency resulting from the two-bead assay format, but this avidityeffect and other peculiarities of the technology complicatedetermination of accurate binding affinities. The IC₅₀ values incompetition AlphaLISA®/AlphaScreen® assays often provide reasonableestimates of the true affinities, but a more rigorous quantitativebinding assay was desired to enable determination of accurate peptideaffinities.

Robust FP Assay for Determining Peptide Affinities for Purified ReceptorECD Complexes.

A fluorescence polarization/anisotropy (FP) peptide-binding assay wasdeveloped using N-terminally FITC-labeled AM(37-52) S45W/Q50W as theprobe, hereafter “FITC-AM W/W.” The probe was custom synthesized andHPLC-purified by RS Synthesis. The large size difference between theMBP-RAMP ECD-CLR ECD fusion proteins (˜65 kDa) and the probe (˜2.5 kDa)ensures a significant anisotropy difference between bound and freestates of the probe. The assay was extensively optimized, and severalcontrols confirmed its validity (data not shown). K_(d) values for theprobe at each of the tethered ECD fusion proteins were determined inequilibrium saturation binding experiments (data not shown), and K_(I)values were determined for unlabeled peptides in equilibrium competitionexperiments (Table 1; FIGS. 2-5). The data were analyzed according toexact analytical equations using non-linear regression curve fitting inGraphPad Prism 5.0 (GraphPad Software) via user-defined equations.CGRP(8-37)NH₂ peptide bound RAMP1-CLR ECD with a 57.5 μM K_(I) in the FPassay, which is in excellent agreement with the 57 μM K_(d) obtained byITC. AM(37-52)NH₂ peptide bound RAMP2-CLR ECD with a 20.4 μM K_(I),which is in good agreement with AlphaLISA® or AlphaScreen® competitionassays (IC₅₀ 5-15 μM). The FP assay indicated that AM Q50W enhancedaffinity more than Q50F and confirmed non-selective nM affinity of theS45W/Q50W double mutant. CGRPmut bound RAMP1-CLR ECD with a K_(I) of 36nM, which is close to the 29 nM IC₅₀ reported for its binding tomembranes of SK-N-MC cells, which express the CGRP receptor.

TABLE 1 Affinity of variant peptides for purified ECD complexes of theCGRP and AM₁ receptors as determined by fluorescence polarization assayRAMP1-CLR RAMP2-CLR ECD (CGRP) ECD (AM₁) Receptor Peptide pK_(I)*; K_(I)(nM) pK_(I); K_(I) (nM) Selectivity AM(37-52)NH₂ scaffold WT<4; >100,000 4.69 ± 0.05; AM₁ over CGRP 20,400 Q50F 4.98 ± 0.03; 10,5005.67 ± 0.06; 5-fold for AM₁ 2140 Q50W 6.30 ± 0.04; 501 6.50 ± 0.06; 316Non-selective K46L No binding No binding K46M No binding No binding D39FNo binding No binding D39K No binding 5.22 ± 0.05; AM₁ 6100 S45W/Q50W7.35 ± 0.06; 44.7 7.09 ± 0.04; Non-selective 81.3 K46L/Q50W 7.15 ± 0.03;70.8 5.78 ± 0.02; 23-fold for CGRP 1660 K46L/Q50W/Y52F 7.50 ± 0.08; 31.64.05 ± 0.17; 2820-fold for CGRP 89,100 D39F/K46L/Q50W 7.10 ± 0.06; 82.25.74 ± 0.07; 22-fold for CGRP 1833 D39F/K46L/Q50W/Y52F 7.61 ± 0.03; 25.04.55 ± 0.08; 1213-fold for CGRP 30,333 S45W/K46L/Q50W/Y52F 8.72 ± 0.11;1.91 5.39 ± 0.05; 2131-fold for CGRP 4070 S45W/K46M/Q50W/Y52F 8.10 ±0.13; 7.94 5.11 ± 0.04; 977-fold for CGRP 7760 S45W/K46W/Q50W/Y52F 7.62± 0.04; 24.0 4.78 ± 0.03; 692-fold for CGRP 16,600 S45T/K46W/Q50W 6.36;439.5 4.90; 12,500 28-fold for CGRP S45T/K46L/Q50W 7.18; 65.6 5.80; 157024-fold for CGRP S45W/K46L/S48G/Q50W/Y52F ND^(#) 5.90; 1250 NDS45W/K46L/I47M/Q50W/Y52F 9.11; 0.78 No binding CGRP (very selective)CGRP CGRP(11-37)NH₂ 4.24 ± 0.10; 57,500 No binding CGRP CGRP(27-37)NH₂scaffold CGRPmut [N31D/S34P/K35F]^(&) 7.44 ± 0.11; 36.3 No binding CGRPN31D/S34P/K35W 8.09 ± 0.03; 8.13 No binding CGRP N31D/S34P/K35F/A36S8.53 ± 0.08; 2.95 <5; >10,000; CGRP over AM₁ N31D/S34P/K35W/A36S 8.88 ±0.10; 1.32 5.61 ± 0.07; 1856-fold for CGRP 2450 N31D/S34P/K35W/A36S/F37Y8.88 ± 0.25; 1.32 6.49 ± 0.09; 324 245-fold for CGRPN31D/V32T/S34P/K35W/A36G 8.16; 6.85 No binding CGRPN31D/V32T/S34P/K35W/A36G/F37Y 7.75; 17.6 4.84; 14,454 821-fold for CGRPN31D/V32T/S34P/K35W/A36S >9; <1.0^(##) 5.56; 2754 ≥2754-fold for CGRP*pK_(I) values are reported as mean ± S.E.M of at least 3 independentexperiments. ^(#)ND; not determinable. ^(&)high-affinity antagonistidentified in Rist, et al., J Med Chem (1998). ^(##)K_(I) in the pMrange beyond what can be reliably measured with our FP assay.

Design and Characterization of AM and CGRP Antagonist Variants withFurther Enhanced Potency and Selectivity.

Superposition of the CGRPmut- and AM-bound structures reveals slightdifferences in the shapes of the pocket occupied by the peptideC-terminal residue. As compared to RAMP1, RAMP2 causes a slight twist ofCLR al relative to the remainder of the ECD that appears to propagate toCLR loop2. The subtle differences in loop2 appear to displace the AM Y52phenyl ring relative to that of CGRPmut F37. It was hypothesized thatthis allosteric effect of RAMPS contributes to selectivity. The singleα-helical turn in AM allows the aliphatic portion of the K46 side chainto contact the CLR “Trp shelf” at the base of the pocket andintramolecularly pack against the AM Y52 phenyl ring. The AM K46 ε-aminogroup is close to RAMP2 E105, and E101 and appears to provide H-bondand/or ionic interactions with these residues; however, mutagenesissuggested that these interactions are insignificant compared to the K46hydrophobic contacts. Several AM variants with K46L or K46M combinedwith the previously identified mutations and/or Y52F to remove the keyH-bond between AM Y52 and RAMP2 E101 were purchased and tested in the FPassay (Table 1; FIGS. 2-3). K46W was also tested even though modelingsuggested a Trp might pack against Y52 without displacing it towardsloop2. The results supported the allostery hypothesis, as several potentAM(37-52)NH₂ variants selective for the CGRP receptor were identified.AM(37-52)NH₂ [S45W/K46L/Q50W/Y52F] had an affinity of ˜2 nM forRAMP1-CLR ECD and was >2000-fold selective. CGRP(27-37)NH₂ variants withaffinity greater than “CGRPmut” (SEQ ID NO:35) were generated byintroducing W at position 35 to provide more hydrophobic contact to CLRloop4 and/or S at position 36 to form an intramolecular H-bond with theD31 backbone carbonyl to stablize the β-turn. These substitutionsgenerated CGRP(27-37)NH₂[N31D/S34P/K35W/A36S] with ˜1 nM affinity forRAMP1-CLR ECD (Table 1; FIGS. 4-5). Inclusion of F37Y yieldedCGRP(27-37)NH₂[N31D/S34P/K35W/A36S/F37Y], which gained significantaffinity for RAMP2-CLR ECD (324 nM K_(I)), but still preferred RAMP1-CLRECD. These results indicated that CGRP and AM can be altered to gainsignificant affinity for the opposite receptor.

ECD or ECD complexes were purified for five of the seven receptorsarising from CLR/CTR and RAMPS: CGRP, AM₁, CTR, AMY₁, and AMY₂ (Table2). Expression and purification of tethered fusions with RAMP3 ECD hasthus far proven difficult. MBP-CTR ECD, MBP-RAMP1-CTR ECD, andMBP-RAMP2-CTR ECD were expressed, purified, and characterized. RAMP1-CLRand RAMP2-CLR ECD were produced in E. coli, but HEK293T cells were usedto produce the CTR-based proteins because N-glycosylation of CTR mayinfluence its ligand binding. CTR ECD, RAMP1-CTR ECD, and RAMP2-CTR ECDreproduced the ligand selectivity of the intact receptors; tetheringRAMP1 to CTR enhanced rAmy and CGRP affinity. Notably, CGRP(27-37)NH₂[N31D/S34P/K35W/A36S] had significantly stronger affinity for RAMP1-CTRthan AM(37-52)NH₂[S45W/K46L/Q50W/Y52F] (FIG. 6), which indicates thatAM-based variants may be more promising as CGRP receptor antagonists formigraines because they are likely to be more selective against AMY₁ thanCGRP-based variants.

TABLE 2 Summary of human receptors for CT family peptides Receptor(designation) Composition Peptide agonist potency CGRP receptor (CGRP)CLR + RAMP1 CGRP > AM, AM2 > Amy Adrenomedullin receptor CLR + RAMP2AM > AM2 >> CGRP > Amy (AM₁) Adrenomedullin receptor CLR + RAMP3 AM >AM2 > CGRP > Amy (AM₂) Calcitonin receptor (CTR) CTR alone CT > Amy,CGRP > AM, AM2 Amylin receptor (AMY₁) CTR + RAMP1 Amy ≈ CT ≈ CGRP >AM2 >> AM Amylin receptor (AMY₂) CTR + RAMP2 Increased Amy affinity, butpoorly defined Amylin receptor (AMY₃) CTR + RAMP3 Amy > CT > CGRP > AM,AM2

This work with purified tethered ECD fusion proteins has indicated thatthey accurately recapitulate the ECD complexes as they exist in theintact receptors, but it is nonetheless useful to characterize theengineered variants at intact receptors in cells. COS-7 cells do notexpress CLR/CTR or RAMPs and thus provide a clean background forpharmacological studies of the heterologously expressed receptors. Theability of AM(37-52)NH₂[S45W/K46L/Q50W/Y52F] to antagonize the CGRP andAM₁ receptors transiently expressed in COS-7 cells was assessed.Forty-eight hours after transfection, the cells were pre-incubated for30 minutes in the absence or presence of 500 nM AM(37-52)NH₂[S45W/K46L/Q50W/Y52F], and then stimulated for 15 minutes withincreasing concentrations of agonist peptide in the absence or presenceof 500 nM antagonist. The cells were lysed, and cAMP was quantitatedusing a LANCE TR-FRET cAMP kit (Perkin-Elmer Waltham, Mass.). Theconcentration-response curves were fit by non-linear regression usingGraphPad Prism 5.0 to a Gaddum/Schild EC₅₀ shift equation with Hill andSchild slopes constrained to 1 to determine pA₂ values (a measure ofantagonist potency). AM(37-52)NH₂[S45W/K46L/Q50W/Y52F] antagonized theCGRP receptor with a pA₂ of ˜8 and did not antagonize the AM₁ receptor(FIGS. 7A,B). This indicates that AM(37-52)NH₂[S45W/K46L/Q50W/Y52F]functions selectively at the intact CGRP receptor in cells and suggestsan affinity of ˜10 nM for the intact receptor, which is close to the ˜2nM K_(I) for the ECD complex (Table 1).

Identification of Potent and Selective Peptides for the CGRP and AMReceptors Using Synthetic Peptide Combinatorial Libraries.

Synthetic peptide combinatorial libraries (SPCL) provide an alternativeto rational design that is a less-biased approach to identify optimizedpeptides from mixtures of thousands to millions of variants. Thepositional scanning-SPCL (PS-SPCL) method can identify optimal aminoacids at a given position considering all possible combinations atseveral other positions. This approach has been used to develop potentpeptides selective for opioid receptors. It was hypothesized that the FPassay could be leveraged to screen AM- and CGRP-based PS-SPCLs forbinding to RAMP1-CLR and RAMP2-CLR ECD to identify potent and selectiveantagonist variants that might have been missed by the rationalapproach.

PS-SPCL for the AM and CGRP Antagonist Fragments and FP-Based Screening.

The number of positions that can be randomized in the PS-SPCL approachdepends on various factors, such as (but not limited to) solubility ofthe peptide mixtures, concentration of mixtures needed to see an effectin the FP assay, etc. Taking these into consideration, 5 of the 16positions in AM(37-52)NH₂[Q50W] (according to SEQ ID NO:53) and 5 of the11 positions in CGRP(27-37)NH₂[S34P, K35W] (according to SEQ ID NO:54)were optimized. Although including AM Q50W and CGRP K35W identified byrational design introduces bias, it was predicted that the non-selectiveaffinity increases conferred by these mutations would permit lowerpeptide concentrations to be used in case of solubility problems. AMpositions S45, K46, 147, S48, and Y52 were chosen as positions forrandomization based on structural considerations. CGRP positions N31,V32, G33, A36, and F37 were chosen for optimization also based onstructural considerations. Each complete PS-SPCL library includes 5“positional libraries” corresponding to the 5 positions to be optimized.Each positional library contains 19 peptide mixtures, wherein thatposition is defined with one of 19 natural amino acids (no cysteine),and the other 4 positions are randomized with an equimolar mixture ofthe 19 amino acids. The 5 AM-based positional libraries were based onSEQ ID NO:53, wherein positions 45, 46, 47, 48, and 52 were defined inconsecutive sequences, and the other four positions in each sequencewere randomized. The 5 CGRP-based positional libraries were based on SEQID NO:54, wherein positions 31, 32, 33, 36, and 37 were defined inconsecutive sequences, and the other four positions in each sequencewere randomized. The total number of peptide mixtures for each libraryis 95 (5*19), and each of the mixtures contains 130,321 unique variantpeptides (19⁴). These custom-synthesized PS-SPCLs were purchased from RSSynthesis (Louisville, Ky.). Individual lyophilized mixtures werereconstituted in 10% DMSO at 10 mg/ml for the AM library or in 25% DMSOat 7.5 mg/ml for the CGRP library. The positional libraries werescreened in the FP competition assay with each AM library mixture at0.05 mg/ml for RAMP1-CLR ECD or 0.5 mg/ml for RAMP2-CLR ECD and eachCGRP library mixture at 0.11 mg/ml for RAMP1-CLR ECD. The CGRP librarywas also screened at RAMP2-CLR ECD, but no significant binding wasobserved (data not shown). The results of the entire screen for the AMlibrary at both the CGRP and AM₁ receptor ECD complexes are shown inFIG. 8, and the results of the entire screen for the CGRP library at theCGRP receptor ECD complex is shown in FIG. 9. These experimentsvalidated the PS-SPCL approach, because W at position 45 and L, W, or Mat position 46 of AM were identified as conferring the strongest bindingto RAMP1-CLR ECD, in agreement with the rational design results.Encouragingly, the results indicated that variants were missed byrational design. For example, R at position 45 of AM conferred strongbinding to RAMP2-CLR ECD but did not enhance binding to RAMP1-CLR ECD.The screen results identify ideal residues at each position that conferstrong affinity when in combination with certain residues at the otherpositions. As more than one good residue was sometimes identified at agiven position, there is some ambiguity in interpreting the screen.Nonetheless, by modeling various peptide sequence combinationsidentified in the screens, additional designs of interest wereidentified. Synthetic peptides corresponding to these designs wereordered and tested in the FP assay for binding to each of the complexes.(FIGS. 2-5). This approach allowed the identification ofAM(37-52)NH₂[S45W, K46L, I47M, Q50W, Y52F] as an extremely high affinity(K_(I) ˜780 pM) antagonist ligand for the CGRP receptor ECD complex withexquisite selectivity, as no binding to the AM₁ receptor ECD complex wasdetected (Table 1). CGRP(27-37)NH₂ [N31D, V32T, S34P, K35W, A36S] wassimilarly identified as an extremely high affinity CGRP receptorantagonist with affinity well into the pM range, such that its affinitycould not be accurately determined by the FP assay (Table 1). Thispeptide was also significantly selective for the CGRP receptor.

Variants of AM and CGRP

The variant peptides of the present disclosure include, but are notlimited to, variants of wild-type human adrenomedullin (AM) and variantfragments thereof, and variants of calcitonin gene-related peptides(α-CGRP and β-CGRP) and variant fragments thereof which bind to at leastone of the CLR:RAMP1, CLR:RAMP2, and CLR:RAMP3 receptor complexes withan affinity characterized by a K_(i) less than about 1 μM, for exampleunder conditions described elsewhere herein. In certain embodiments, thevariants have higher receptor affinity and increased selectivity thanwild type AM and/or wild type α-CGRP, and/or wild type β-CGRP. Thevariants, in general, are C-terminally amidated. Any of the variantpeptides disclosed herein can be used with a pharmaceutically-acceptablecarrier in a composition, for example for use in treatment of theconditions and diseases described elsewhere herein.

In at least certain embodiments, amino acids 1-12 of AM can be deletedwithout affecting binding activity of the remaining AM fragment. In atleast certain embodiments, approximately the N-terminal half of the13-52 peptide fragment of SEQ ID NO:1 interacts with the receptor7-transmembrane (7TM) domain and the C-terminal portion interacts withthe receptor extracellular domain (ECD). The N-terminal portion (e.g.,amino acids 13-21) causes activation of the receptor 7TM domain; thusthe 13-52 peptide has agonistic activity. Antagonistic versions (whichbind to one or more of the CLR:RAMP1-3 receptor complexes but which donot activate the receptor complex) can be generated by deleting theN-terminal portion comprising amino acids 13-21. Thus AM variants basedon amino acids 22-52 of SEQ ID NO:1 or, for example, AM fragmentsconsisting of amino acids 37-52 of AM (DKDNVAPRSKISPQGY—SEQ ID NO:4) andvariants thereof, have an antagonistic effect on the correspondingCLR:RAMP receptor complex.

Any mutation in a C-terminal portion in AM that causes higher receptoraffinity and/or selectivity in a shortened antagonist fragment will alsogive rise to an agonistic peptide with a higher affinity (and therebyhigher potency) when incorporated into the full length AM peptide (SEQID NO:1) or into an agonistic fragment thereof (e.g., amino acids 13-52of SEQ ID NO:1).

Similarly, any mutation in a C-terminal portion of CGRP that causeshigher receptor affinity and/or selectivity in a shortened antagonistfragment (e.g., amino acids 8-37) will also give rise to an agonisticpeptide with a higher affinity (and thereby higher potency) whenincorporated into the full length CGRP peptide (37 amino acids) or intoan agonistic fragment thereof (in CGRP peptides, the N terminal 1-7amino acid portion activates the receptor 7TM domain; thus the 8-37peptide is an antagonist). CGRP peptide fragment FVPTNVGSKAF-NH₂ (SEQ IDNO:34) is a C-terminal binding portion having antagonistic activityagainst the cognate receptor complex. FVPTDVGPFAF-NH₂ (SEQ ID NO: 35) isan antagonistic mutant of this peptide fragment, having high affinityfor CGRP receptor (see Rist et al. (J Med Chem. (1998) 41(1):117-23)),but it also has higher affinity for the AM₁ receptor.

The embodiments of the present disclosure include, but are not limitedto, variants and variant fragments of wild-type human adrenomedullin(AM) and/or calcitonin gene-related peptide (CGRP) peptide which havehigher affinity than the wild type peptide for at least one of, two of,or all three of the AM₁, AM₂, and CGRP receptors, wherein higheraffinity means at least 50-fold, at least 100-fold, at least 200-fold,at least 300-fold, at least 400-fold, at least 500-fold, at least600-fold, at least 700-fold, at least 800-fold, at least 900-fold, atleast 1000-fold, at least 1100-fold, at least 1200-fold, at least1300-fold, at least 1400-fold, at least 1500-fold, at least 1600-fold,at least 1700-fold, at least 1800-fold, at least 1900-fold, at least2000-fold, at least 2500-fold, at least 3000-fold, at least 3500-fold,at least 4000-fold, at least 4500-fold, at least 5000-fold, at least5500-fold, at least 6000-fold, at least 6500-fold, at least 7000-fold,at least 7500-fold, at least 8000-fold, at least 8500-fold, at least9000-fold, at least 9500-fold, at least 10000-fold, at least 15000-fold,or at least 20000-fold, or greater, affinity, for at least one of, twoof, or all three of the AM₁, AM₂, and CGRP receptors. In certainembodiments, the variant peptides bind only to the ECD complex of CLRand the associated RAMP portion of the receptor (wherein the variant isa receptor complex antagonist), or to both the ECD complex and the 7TMportion of CLR (wherein the variant peptide is a receptor complexagonist). The variant peptides of the present disclosure may haveaffinity K_(i)'s which are <1 μM, <750 nM, <500 nM, <400 nM, <300 nM,<200 nM, <100 nM, <50 nM, <10 nM, <1nM, <100 pM, <10 pM, <5 pM, <2 pM,or <1 pM when binding to the receptor complex CLR:RAMP1, CLR:RAMP2,and/or CLR:RAMP3 is measured.

The AM and CGRP variant peptides of the present disclosure have, but arenot limited to, one of at least two effects: (1) an agonistic effect onthe corresponding CLR:RAMP1-3 receptor complex, and (2) an antagonisticeffect against the corresponding CLR:RAMP1-3 receptor complex. Theseeffects lead to particular utilities. For example, AM peptides havingagonistic effects (e.g., peptides having both N-terminal and C-terminalbinding portions) can be used therapeutically for treatment of, forexample, heart failure, acute myocardial infarction, pulmonaryhypertension, pre-eclampsia, sepsis, lymphedema, lymphangectasia, andfertility (e.g., implantation during in vitro fertilization), and otherconditions discussed elsewhere herein. AM peptides having antagonisticeffects (e.g., peptides having only a C-terminal binding portion) can beused therapeutically for treatment of, for example, cancers as describedelsewhere herein. CGRP peptides having antagonistic effects (e.g.,peptides having only a C-terminal binding portion) can be usedtherapeutically for treatment of, for example, cancers, migraineheadaches, and other conditions described elsewhere herein.

In certain embodiments, the present disclosure includes a method oftreating a subject for a condition regulated by a calcitoninreceptor-like receptor-receptor activity-modifying protein (CLR:RAMP)receptor complex, comprising the step of administering to a subject inneed of such therapy, an effective amount of a variant peptide of atleast one of adrenomedullin (AM), calcitonin gene-related peptide alpha(αCGRP), and calcitonin gene-related peptide beta (βCGRP). The CLR:RAMPreceptor complex is one or more of CLR:RAMP1, CLR:RAMP2, and CLR:RAMP3.

An effective amount of a variant peptide composition of the presentdisclosure will generally contain sufficient active substance to deliverfrom about 0.01 μg/kg to about 100 mg/kg (weight of activesubstance/body weight of the subject). Particularly, the compositionwill deliver about 0.1 μg/kg to about 50 mg/kg, and more particularlyabout 1 μg/kg to about 20 mg/kg.

Practice of the methods of the present disclosure may compriseadministering to a subject an effective amount of the variant peptide inany suitable systemic and/or local formulation in an amount effective todeliver the dosages listed above. The dosage can be administered, forexample but not by way of limitation, on a one-time basis, oradministered at multiple times (for example but not by way oflimitation, from one to five times per day, or once or twice per week),or continuously via a venous drip, depending on the desired therapeuticeffect. In one non-limiting example of a therapeutic method of thepresent disclosure, the variant peptide compound is provided in an IVinfusion in the range of from about 0.01 mg/kg to about 10 mg/kg of bodyweight once a day.

Administration of the peptide compound used in the pharmaceuticalcomposition or to practice the method of the present disclosure can becarried out in a variety of conventional ways, such as, but not limitedto, orally, by inhalation, rectally, topically, nasally, or bycutaneous, subcutaneous, intraperitoneal, vaginal, or intravenousinjection. Oral formulations may be formulated such that the variantpeptide passes through a portion of the digestive system before beingreleased, for example it may not be released until reaching the smallintestine, or the colon.

When an effective amount of the variant peptide is administered orally,it may be in the form of a solid or a liquid preparation such as (butnot limited to) capsules, pills, tablets, lozenges, melts, powders,suspensions, solutions, elixirs, or emulsions. Solid unit dosage formscan be capsules of the ordinary gelatin type containing, for example,surfactants, lubricants, and inert fillers such as lactose, sucrose, andcornstarch, or the dosage forms can be sustained release preparations.The pharmaceutical composition may contain a solid carrier, such as agelatin or an adjuvant. The tablet, capsule, and powder may contain fromabout 0.05% to about 95% of the active substance compound by dry weight.When administered in liquid form, a liquid carrier such as water,petroleum, oils of animal or plant origin such as peanut oil, mineraloil, soybean oil, or sesame oil, or synthetic oils may be added. Theliquid form of the pharmaceutical composition may further containphysiological saline solution, dextrose or other saccharide solution, orglycols such as ethylene glycol, propylene glycol, or polyethyleneglycol. When administered in liquid form, the pharmaceutical compositionparticularly contains from about 0.005% to about 95% by weight of theactive substance. For example, a dose of about 10 mg to about 1000 mgonce or twice a day could be administered orally.

In another embodiment, the variant peptides of the present disclosurecan be tableted with conventional tablet bases such as (but not limitedto) lactose, sucrose, and cornstarch in combination with binders, suchas (but not limited to) acacia, cornstarch, or gelatin, disintegratingagents such as (but not limited to) potato starch or alginic acid, and alubricant such as (but not limited to) stearic acid or magnesiumstearate. Liquid preparations are prepared by dissolving the peptidecompound in an aqueous or non-aqueous pharmaceutically acceptablesolvent which may also contain suspending agents, sweetening agents,flavoring agents, and preservative agents as are known in the art.

For parenteral administration, for example, the peptide compounds may bedissolved in a physiologically acceptable pharmaceutical carrier andadministered as either a solution or a suspension. Illustrative ofsuitable non-limiting pharmaceutical carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative, or synthetic origin. The pharmaceutical carrier may alsocontain preservatives and buffers as are known in the art.

When an effective amount of the variant peptide is administered byintravenous, cutaneous, or subcutaneous injection, the compound isparticularly in the form of a pyrogen-free, parenterally acceptableaqueous solution or suspension. The preparation of such parenterallyacceptable solutions, having due regard to pH, isotonicity, stability,and the like, is well within the skill in the art. A particularpharmaceutical composition for intravenous, cutaneous, or subcutaneousinjection may contain, in addition to the peptide compound, an isotonicvehicle such as (but not limited to) Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, or other vehicle as known in the art. Thepharmaceutical compositions of the present disclosure may also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art.

As noted, particular amounts and modes of administration can bedetermined by one skilled in the art. One skilled in the art ofpreparing formulations can readily select the proper form and mode ofadministration, depending upon the particular characteristics of thepeptide compound selected, the condition to be treated, the stage of thecondition, and other relevant circumstances using formulation technologyknown in the art, described, for example, in Remington: The Science andPractice of Pharmacy, 21^(st) ed.

Additional pharmaceutical methods may be employed to control theduration of action of the variant peptide. Increased half-life and/orcontrolled release preparations may be achieved through the use ofproteins or polymers to conjugate, complex with, and/or absorb thepeptide as discussed previously herein. The controlled delivery and/orincreased half-life may be achieved by selecting appropriatemacromolecules (for example but not by way of limitation,polysaccharides, polyesters, polyamino acids, homopolymers, polyvinylpyrrolidone, ethylenevinylacetate, methylcellulose, orcarboxymethylcellulose, and acrylamides such as N-(2-hydroxypropyl)methacrylamide), and the appropriate concentration of macromolecules aswell as the methods of incorporation, in order to control release.

Another possible method useful in controlling the duration of action ofthe peptide compound by controlled release preparations and half-life isincorporation of the peptide compound or its functional derivatives intoparticles of a polymeric material such as (but not limited to)polyesters, polyamides, polyamino acids, hydrogels, poly(lactic acid),ethylene vinylacetate copolymers, copolymer micelles of, for example,polyethylene glycol (PEG) and poly(1-aspartamide).

It is also possible to entrap the peptide compounds in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization (for example, hydroxymethylcellulose orgelatine-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules), or in macroemulsions. Such techniques are well known topersons having ordinary skill in the art.

When the variant peptide or peptide compound is to be used as aninjectable material, it can be formulated into a conventional injectablecarrier. Suitable carriers include (but not limited to) biocompatibleand pharmaceutically acceptable phosphate buffered saline solutions,which are particularly isotonic.

For reconstitution of a lyophilized product in accordance with thepresent disclosure, one may employ a sterile diluent, which may containmaterials generally recognized for approximating physiologicalconditions and/or as required by governmental regulation. In thisrespect, the sterile diluent may contain a buffering agent to obtain aphysiologically acceptable pH, such as sodium chloride, saline,phosphate-buffered saline, and/or other substances which arephysiologically acceptable and/or safe for use. In general, the materialfor intravenous injection in humans should conform to regulationsestablished by the US Food and Drug Administration, which are availableto those in the field. The pharmaceutical composition may also be in theform of an aqueous solution containing many of the same substances asdescribed above for the reconstitution of a lyophilized product.

The variant peptides can also be administered as a pharmaceuticallyacceptable acid- or base-addition salt, formed by reaction withinorganic acids such as (but not limited to) hydrochloric acid,hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, and phosphoric acid, and organic acids such as (but notlimited to) formic acid, acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, and fumaric acid, or by reaction with an inorganic basesuch as (but not limited to) sodium hydroxide, ammonium hydroxide,potassium hydroxide, and organic bases such as (but not limited to)mono-, di-, trialkyl and aryl amines, and substituted ethanolamines.

In certain embodiments, the present disclosure includes a variantpeptide composition including: at least one variant peptide coupleddirectly or indirectly to a carrier molecule, wherein the at least onevariant peptide is from 10 to 70 amino acids in length.

The compounds, conjugates, compositions, and methods of production andapplication of the variant peptides described herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the present disclosure has been described inconnection with certain embodiments so that aspects thereof may be morefully understood and appreciated, it is not intended that the presentdisclosure be limited to these particular embodiments. On the contrary,it is intended that all alternatives, modifications and equivalents areincluded within the scope of the present disclosure. Thus the examplesdescribed above, which include particular embodiments, will serve toillustrate the practice of the present disclosure, it being understoodthat the particulars shown are by way of example and for purposes ofillustrative discussion of particular embodiments only and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of procedures as well as of theprinciples and conceptual aspects of the presently disclosed methods andcompositions. Changes may be made in the formulation of the variouscompositions described herein, the methods described herein or in thesteps or the sequence of steps of the methods described herein withoutdeparting from the spirit and scope of the present disclosure.

1. A variant peptide having binding affinity for at least one receptorcomplex of the group consisting of calcitonin receptor-likereceptor-receptor activity-modifying protein 1 (CLR:RAMP1), calcitoninreceptor-like receptor-receptor activity-modifying protein 2(CLR:RAMP2), and calcitonin receptor-like receptor-receptoractivity-modifying protein 3 (CLR:RAMP3), the variant peptide having anaffinity K_(i) for the receptor complex of less than 1 μM, and thevariant peptide having a structure as represented by Formula I (SEQ IDNO:49):X₁-K-X₃-N-V-A-P-R-X₉-X₁₀-X₁₁-X₁₂-P-X₁₄-G-X₁₆   (I) wherein: X₁ is A, D,V, L, or I, X₃ is D, F, M, Y, V, I, E, K, W, L, or R, X₉ is S, W, Y, F,R, T, or L, X₁₀ is K, G, L, M, Y, A, W, or F, X₁₁ is I, L, or M, X₁₂ isS, T, or G, X₁₄ is Q, F, Y, W, or H, and X₁₆ is Y or F, wherein the Y orF is amidated; with the proviso that the variant peptide does notinclude amino acids 37-52 of SEQ ID NO:1 in a contiguous sequence; andwherein the variant peptide is optionally extended at the N-terminus byall or any portion of amino acids 1-36 of SEQ ID NO:1, by all or anyportion of amino acids 1-22 of SEQ ID NO:31, by all or any portion ofamino acids 1-22 of SEQ ID NO:32, or by all or any portion of aminoacids 1-24 of SEQ ID NO:47.
 2. The variant peptide of claim 1, whereinthe variant peptide is bound to a carrier molecule, and optionally isbound to the carrier molecule by a linker molecule.
 3. (canceled)
 4. Thevariant peptide of claim 1, further defined as consisting of up to 70amino acids.
 5. The variant peptide of claim 1, further defined ashaving agonistic activity for the at least one receptor complex.
 6. Thevariant peptide of claim 1, further defined as having antagonisticactivity for the at least one receptor complex.
 7. The variant peptideof claim 1, wherein the affinity K_(i) is less than about 500 nM. 8-14.(canceled)
 15. A variant peptide having binding affinity for at leastone receptor complex of the group consisting of CLR:RAMP1, CLR:RAMP2,and CLR:RAMP3, the variant peptide having an affinity K_(i) for thereceptor complex of less than 1 μM, and the variant peptide having astructure as represented by Formula III (SEQ ID NO:51):F-V-P-T-X₅-X₆-G-X₈-X₉-X₁₀-X₁₁   (III) wherein: X₅ is N, D, or W, X₆ isV, T, or W, X₈ is P or S, X₉ is W, Y, or H, X₁₀ is A, S, or G, X₁₁ is For Y, wherein the F or Y is amidated; wherein the peptide is optionallyextended at the N-terminus by all or any portion of amino acids 1-41 ofSEQ ID NO:1, all or any portion of amino acids 1-26 of SEQ ID NO:31, allor any portion of amino acids 1-26 of SEQ ID NO:32, or all or anyportion of amino acids 1-29 of SEQ ID NO:47.
 16. The variant peptide ofclaim 15, wherein the variant peptide is bound to a carrier molecule,and optionally is bound to the carrier molecule by a linker molecule.17. (canceled)
 18. The variant peptide of claim 15, further defined asconsisting of up to 70 amino acids.
 19. The variant peptide of claim 15,further defined as having agonistic activity for the at least onereceptor complex.
 20. The variant peptide of claim 15, further definedas having antagonistic activity for the at least one receptor complex.21. The variant peptide of claim 15, wherein the affinity K_(i) is lessthan about 500 nM. 22-28. (canceled)
 29. A method of treating a subjectfor a condition regulated by a calcitonin receptor-likereceptor-receptor activity-modifying protein (CLR:RAMP) receptorcomplex, comprising the step of: administering an effective amount of avariant peptide of at least one of adrenomedullin (AM), calcitoningene-related peptide alpha (αCGRP), and calcitonin gene-related peptidebeta (βCGRP) to a subject in need of such therapy.
 30. The method ofclaim 29, wherein the CLR:RAMP receptor complex is CLR: RAMP1 .
 31. Themethod of claim 29, wherein the CLR:RAMP receptor complex is CLR:RAMP2.32. The method of claim 29, wherein the CLR:RAMP receptor complex isCLR:RAMP3.
 33. The method of claim 29, wherein the variant peptidecomprises a structure as represented by Formula I (SEQ ID NO:49):X₁-K-X₃-N-V-A-P-R-X₉-X₁₀-X₁₁-X₁₂-P-X₁₄-G-X₁₆   (I) wherein: X₁ is A, D,V, L, or I, X₃ is D, F, M, Y, V, I, E, K, W, L, or R, X₉ is S, W, Y, F,R, T, or L, X₁₀ is K, G, L, M, Y, A, W, or F, X₁₁ is I, L, or M, X₁₂ isS, T, or G, X₁₄ is Q, F, Y, W, or H, and X₁₆ is Y or F, wherein the Y orF is amidated; with the proviso that the variant peptide does notinclude amino acids 37-52 of SEQ ID NO:1 in a contiguous sequence; andwherein the variant peptide is optionally extended at the N-terminus byall or any portion of amino acids 1-36 of SEQ ID NO:1, by all or anyportion of amino acids 1-22 of SEQ ID NO:31, by all or any portion ofamino acids 1-22 of SEQ ID NO:32, or by all or any portion of aminoacids 1-24 of SEQ ID NO:47.
 34. The method of claim 29, wherein thevariant peptide comprises a structure as represented by Formula II (SEQID NO:50):A-P-R-X₄-X₅-X₆-X₇-P-X₉-G-X₁₁   (II) wherein: X₄ is S , W, Y, F, R, T, orL, X₅ is K, G, L, M, Y, A, W, or F, X₆ is I, L, or M, X₇ is S, T, or G,X₉ is Q, F, Y, W, or H, and X₁₁ is Y or F, wherein the Y or F isamidated; with the proviso that the variant peptide does not includeamino acids 42-52 of SEQ ID NO:1 in a contiguous sequence; and whereinthe peptide is optionally extended at the N-terminus by all or anyportion of amino acids 1-41 of SEQ ID NO:1, all or any portion of aminoacids 1-26 of SEQ ID NO:31, all or any portion of amino acids 1-26 ofSEQ ID NO:32, or all or any portion of amino acids 1-29 of SEQ ID NO:47.35. The method of claim 29, wherein the variant peptide comprises astructure as represented by Formula III (SEQ ID NO:51):F-V-P-T-X₅-X₆-G-X₈-X₉-X₁₀-X₁₁   (III) wherein: X₅ is N, D, or W, X₆ isV, T, or W, X₈ is P or S, X₉ is W, Y, or H, X₁₀ is A, S, or G, X₁₁ is For Y, wherein the F or Y is amidated; wherein the peptide is optionallyextended at the N-terminus by all or any portion of amino acids 1-41 ofSEQ ID NO:1, all or any portion of amino acids 1-26 of SEQ ID NO:31, allor any portion of amino acids 1-26 of SEQ ID NO:32, or all or anyportion of amino acids 1-29 of SEQ ID NO:47.
 36. The method of claim 29,wherein the variant peptide comprises a structure as represented byFormula IV (SEQ ID NO:52):T-X₂-X₃-G-X₅-X₆-X₇-X₈   (IV) wherein: X₂ is N, D, or W, X₃ is V, T, orW, X₅ is P or S, X₆ is W, Y, or H, X₇ is A, S, or G, X₈ is F or Y,wherein the F or Y is amidated; wherein the peptide is optionallyextended at the N-terminus by all or any portion of amino acids 1-44 ofSEQ ID NO:1, all or any portion of amino acids 1-29 of SEQ ID NO:31, allor any portion of amino acids 1-29 of SEQ ID NO:32, or all or anyportion of amino acids 1-32 of SEQ ID NO:47. 37-39. (canceled)